cloning and expression of pluripotent factors around the time of
TRANSCRIPT
CLONING AND EXPRESSION OF PLURIPOTENT FACTORS AROUND THE TIME OF GASTRULATION IN THE PORCINE CONCEPTUS
by
DOUGLAS ROBERT EBORN
B.S., Utah State University, 2000 M.S., Kansas State University 2005
AN ABSTRACT OF A DISSERTATION
submitted in partial fulfillment of the requirements for the degree
DOCTOR OF PHILOSOPHY
Department of Animal Sciences and Industry College of Agriculture
KANSAS STATE UNIVERSITY Manhattan, Kansas
2008
Abstract
Early in embryonic development a series of events occur whereby pluripotent cells
undergo differentiation to give rise to the three germ layers and extraembryonic tissues of the
developing conceptus. Nanog, Sox-2, and Oct-4 genes have been identified as having key roles
in maintaining pluripotency in undifferentiated human and mouse cells but recent evidence
suggests they may have different roles in farm animals. We cloned the coding sequence for
porcine Nanog including 452 base pairs of the Nanog promoter, and partial coding sequences of
Oct-4 and Sox-2. Embryos were flushed from sows 10, 12, 15, and 17 days post insemination.
RNA was isolated from whole d-10 and -12 conceptuses, d-15 embryonic disk, distal and
proximal extraembryonic tissue, and d-17 embryonic disk, distal and proximal extraembryonic
tissue, and allantois for real-time PCR. RNA from d-40 maternal myometrium and
endometrium, fetal placenta, and liver were also used in real-time PCR. The homeodomain and
c-terminal tryptophan repeats are highly conserved in porcine Nanog compared to the mouse,
human and bovine. In the promoter, the highly conserved Octamer and Sox binding sequences
are also present. The Nanog expression pattern was different when compared to Oct-4 and Sox-
2. Day-40 tissues demonstrated the highest expression including endometrium (7 fold) fetal liver
(27 fold), placenta (40 fold) and myometrium (72 fold) when compared to day 15 distal
extraembryonic tissue. Oct-4 and Sox-2 expression was lowest in d-40 tissues except for fetal
liver which was 20 and 71 fold, respectively, higher than endometrium. Oct-4 levels were
consistent in d-10, -12, and -15 conceptuses and disk but dropped 3 fold in d-17 disk. On the
other hand, Sox-2 was upregulated a 1000 fold in the d-15 disk and 2000 fold in the d-17 disk
when compared to the d-12 conceptus. Nanog may have other roles in than maintenance of
pluripotency including a possible role in multipotent or progenitor stem cells. Expression of all 3
markers in fetal liver suggests a more primitive cell type is present such as hematopoietic stem
cells.
CLONING AND EXPRESSION OF PLURIPOTENT FACTORS AROUND THE TIME OF GASTRULATION IN THE PORCINE CONCEPTUS
by
DOUGLAS ROBERT EBORN
B.S., Utah State University, 2000 M.S., Kansas State University 2005
A DISSERTATION
submitted in partial fulfillment of the requirements for the degree
DOCTOR OF PHILOSOPHY
Department of Animal Sciences and Industry College of Agriculture
KANSAS STATE UNIVERSITY Manhattan, Kansas
2008
Approved by:
Major Professor David M. Grieger
Abstract
Early in embryonic development a series of events occur whereby pluripotent cells
undergo differentiation to give rise to the three germ layers and extraembryonic tissues of the
developing conceptus. Nanog, Sox-2, and Oct-4 genes have been identified as having key roles
in maintaining pluripotency in undifferentiated human and mouse cells but recent evidence
suggests they may have different roles in farm animals. We cloned the coding sequence for
porcine Nanog including 452 base pairs of the Nanog promoter, and partial coding sequences of
Oct-4 and Sox-2. Embryos were flushed from sows 10, 12, 15, and 17 days post insemination.
RNA was isolated from whole d-10 and -12 conceptuses, d-15 embryonic disk, distal and
proximal extraembryonic tissue, and d-17 embryonic disk, distal and proximal extraembryonic
tissue, and allantois for real-time PCR. RNA from d-40 maternal myometrium and
endometrium, fetal placenta, and liver were also used in real-time PCR. The homeodomain and
c-terminal tryptophan repeats are highly conserved in porcine Nanog compared to the mouse,
human and bovine. In the promoter, the highly conserved Octamer and Sox binding sequences
are also present. The Nanog expression pattern was different when compared to Oct-4 and Sox-
2. Day-40 tissues demonstrated the highest expression including endometrium (7 fold) fetal liver
(27 fold), placenta (40 fold) and myometrium (72 fold) when compared to day 15 distal
extraembryonic tissue. Oct-4 and Sox-2 expression was lowest in d-40 tissues except for fetal
liver which was 20 and 71 fold, respectively, higher than endometrium. Oct-4 levels were
consistent in d-10, -12, and -15 conceptuses and disk but dropped 3 fold in d-17 disk. On the
other hand, Sox-2 was upregulated a 1000 fold in the d-15 disk and 2000 fold in the d-17 disk
when compared to the d-12 conceptus. Nanog may have other roles in than maintenance of
pluripotency including a possible role in multipotent or progenitor stem cells. Expression of all 3
markers in fetal liver suggests a more primitive cell type is present such as hematopoietic stem
cells.
v
Table of Contents
List of Figures ............................................................................................................................... vii
List of Tables ............................................................................................................................... viii
Dedication ...................................................................................................................................... ix
CHAPTER 1 - Literature Review................................................................................................... 1
Gene Characterisitcs of Nanog, Oct-4 and Sox-2....................................................................... 1
Nanog...................................................................................................................................... 1
Oct-4 ....................................................................................................................................... 3
Sox-2 ....................................................................................................................................... 4
Gene Regulation...................................................................................................................... 4
Gene Expression ..................................................................................................................... 6
Literature Cited ........................................................................................................................... 9
CHAPTER 2 - Cloning and Expression of Pluripotent Factors Around the Time of Gastrulation
in the Porcine Conceptus ....................................................................................................... 13
Introduction............................................................................................................................... 13
Material and Methods ............................................................................................................... 14
Tissue Collection .................................................................................................................. 14
RNA and Protein Isolation.................................................................................................... 14
Gene Cloning ........................................................................................................................ 15
Real-time PCR ...................................................................................................................... 16
Northern Blotting .................................................................................................................. 18
Western Blotting ................................................................................................................... 19
Results....................................................................................................................................... 20
Cloning.................................................................................................................................. 20
Real-time PCR ...................................................................................................................... 21
Northern Blots....................................................................................................................... 21
Western Blots........................................................................................................................ 22
Discussion................................................................................................................................. 22
Literature Cited ......................................................................................................................... 25
vi
Figures and Tables .................................................................................................................... 29
CHAPTER 3 - Timed Insemination of Beef Heifers using the 7-11 Synch Protocol................... 43
Abstract..................................................................................................................................... 43
Introduction............................................................................................................................... 43
Material and Methods ............................................................................................................... 44
Experiment 1 ......................................................................................................................... 44
Experiment 2 ......................................................................................................................... 45
Semen Analysis..................................................................................................................... 46
Statistical Analysis................................................................................................................ 46
Results....................................................................................................................................... 47
Experiment 1 ......................................................................................................................... 47
Experiment 2 ......................................................................................................................... 47
Discussion................................................................................................................................. 48
Cyclicity ................................................................................................................................ 48
Synchronization and Pregnancy Rates.................................................................................. 48
Literature Cited ......................................................................................................................... 51
Figures and Tables .................................................................................................................... 53
vii
List of Figures
Figure 1 Porcine Nanog Nucleotide and Amino Acid Sequence................................................ 29
Figure 2 Porcine Nanog amino acid alignment with the Human, Bovine and Mouse proteins... 30
Figure 3 Porcine Nanog Promoter ............................................................................................... 31
Figure 4 Porcine Nanog Sequence Alignments with Bovine, Human and Mouse ...................... 32
Figure 5 Partial Porcine Oct-4 Sequence ..................................................................................... 33
Figure 6 Partial Porcine Sox-2 Sequence..................................................................................... 34
Figure 7 Relative Nanog Expression in the Early Porcine Conceptus, Fetal Liver, Placenta, and
Maternal Endometrium and Myometrium ............................................................................ 37
Figure 8 Relative Oct-4 Expression in the Early Porcine Conceptus, Fetal Liver, Placenta, and
Maternal Endo- and Myometrium ........................................................................................ 38
Figure 9 Relative Sox-2 Expression in the Early Porcine Conceptus, Fetal Liver, Placenta, and
Maternal Endo- and Myometrium ........................................................................................ 39
Figure 10 Northern Blot of Porcine Sox-2................................................................................... 40
Figure 11 Northern Blots of Porcine β-Actin .............................................................................. 41
Figure 12 Western Blot of Porcine Nanog................................................................................... 42
Figure 13. Experiment 1 treatment schedule for heifers assigned to the 7-11 Synch and 7-11
CIDR protocols. .................................................................................................................... 53
Figure 14. Experiment 1 treatment schedule for heifers assigned to the 7-11 Synch and 7 Synch
protocols................................................................................................................................ 54
viii
List of Tables
Table 1 Adjusted Threshold Means for Nanog, Sox-2, and Oct-4 by Tissue.............................. 35
Table 2 Comparison of 7-11 Synch using MGA (7-11 Synch) or CIDR (7-11 CIDR) on different
reproductive traits of heifers (Exp. 1) ................................................................................... 55
Table 3 Comparison of 7-11 Synch with (7-11 Synch) or without (7 Synch) GnRH on d 11 of
treatment on different reproductive traits of heifers (Exp. 2) ............................................... 56
Table 4 Puberty status before treatment, overall puberty status after treatment, heifers with serum
progesterone (P4) concentrations ≥1 ng/ml, and d 18 serum progesterone concentration of
heifers by location................................................................................................................. 57
Table 5 Pregnancy rates to 48 hr timed AI ................................................................................... 58
Table 6 Puberty status before treatment, overall puberty status after treatment, heifers with serum
progesterone (P4) concentrations ≥1 ng/ml, and d 18 serum progesterone concentration of
heifers by location................................................................................................................. 59
Table 7 Pregnancy rates to 54 hr timed AI ................................................................................... 60
Table 8 Pregnancy rate of heifers with low P4 on d 18 ............................................................... 61
Table 9 P4 status of heifers that did not conceive ....................................................................... 62
Table 10 Semen Analysis.............................................................................................................. 63
ix
Dedication
To my family; Mary Ann, Shaun, Megan, Camille, Elaine, and Julie Ann for their
example of patience, long-suffering, faith, and love.
1
CHAPTER 1 - Literature Review
At fertilization, an oocyte and sperm merge into a single totipotent cell, or zygote, that
has the ability to form every cell type of the conceptus. Eventually, during the process of
cleavage, cells begin to differentiate. The first visual evidence of differentiation is in the
formation of a blastocyst with the trophoblast surrounding the inner cell mass and blastocoele
cavity. The inner cell mass is comprised of undifferentiated cells that will form the embryo
proper and is the source of embryonic stem cells grown in culture. Embryonic stem cells have
made an invaluable contribution to our early understanding of how gene regulation maintains
pluripotency and events that lead to differentiation. Nanog, Sox-2 and Oct-4 are three
transcription factors that are important for the development of the early mouse embryo and
maintaining pluripotency in embryonic stem cells. This review will begin to characterize each
factor in the pig, describe their expression patterns in embryonic development, and address their
regulation and roles in regulation of other genes.
Gene Characterisitcs of Nanog, Oct-4 and Sox-2
Nanog
Named for the mythological Celtic land Tir nan Og (land of the ever young), Nanog is
homeobox transcription factor expressed in pluripotent cells such as embryonic stem (ES), germ
(EG) and carcinoma (EC) as well as the pluripotent cells of the developing embryo. It was first
identified by Wang et al., (2003) who identified a transcription factor expressed in the early
developing mouse embryo and ES cells. It was termed ENK (early embryo specific NK) due to
the presence of a homeodomain that shared 50% identity with other NK-2 family proteins. Later
two independent groups identified the same transcription factor which could maintain
pluripotency in ES cells independent of leukemia inhibitory factor (LIF) stimulation and named
it Nanog (Chambers et al., 2003; Mitsui et al., 2003).
Mouse
Located on chromosome 6, Nanog (GeneID: 71950) is comprised of 4 exons that encodes
a 305 amino acid protein from a transcript of 1356 nucleotides (Accession NM_028016.2). This
2
RefSeq entry replaces an earlier version of Nanog that was 2188 nucleotides in length
(Accession NM_028016.1). Found within the 3’ untranslated region is a B2 repetitive element
that may contribute to gene regulation (Chambers et al., 2003). The Nanog protein can be
broken down into three domains, the N-terminal, homeodomain, and C-terminal. The
homeodomain is contained within residues 98-155 and is unique as the family of NK-2
homeoproteins share the most identity with the homeodomain of Nanog but it’s less than 50%
(Mitsui et al., 2003). N-terminal to the homeodomain is a serine-rich motif and at the C-
terminus is a tryptophan at every fifth position repeated ten times (Chambers et al., 2003; Mitsui
et al., 2003). Both N- and C-terminal domains have transactivation abilities when fused to the
binding domain of the yeast transcription factor, Gal4, but the C-terminal is 7 times more active
than the N-terminus (Pan and Pei, 2003).
Human
Human NANOG (GeneID: 79923) is located on human chromosome 12 and encodes a
2098 nucleotide transcript (Accession NM_024865.2). The protein is also comprised of 305
amino acids with the homeodomain spanning from residues 98-154. Overall amino acid identity
with the mouse is 57.7% but the homeobox is 87.5% similar. The trytophan repeats are
conserved in the human except for a short deletion and a glutamine replacing a tryptophan at
position 211 (Chambers et al., 2003). A high number of pseudogenes have been reported for
human NANOG which include one tandem duplicate and ten processed pseudogenes and this is
much higher than 2 pseudogenes found in the mouse genome (Booth and Holland, 2004). Unlike
the mouse protein where both the N- and C-terminal domains show transactivation activity, only
the C-terminal domain of human Nanog was shown to have activity (Oh et al., 2005).
Farm Animals
Bovine (GeneID: 538951) and porcine (GeneID: 595109) NANOG are located on
chromosome 5. The bovine transcript (Accession NM_001025344.1) is generated from five
exons and is 1644 nucleotides long resulting in a 300 amino acid protein. Amino acid identity
between bovine and the mouse and human is 58.2 and 69.3% overall and 87.9 and 94.6% within
the homeobox respectively. The full-length transcript has not been published for porcine Nanog.
3
Oct-4
Oct-4 belongs to a large family of transcription factors containing a DNA-binding
domain called POU. The domain was named due to sequence similarity among 3 mammalian
transcription factors Pit-1, Oct1 and Oct2, and UNC-86 (Herr et al., 1988). The POU domain is
unique in that it is comprised of two subdomains, the POU-specific (POUS) or POU domain and
the POU homeodomain (POUHD) connected by a linker. The two domains act independently of
each other (Herr and Cleary, 1995). When binding to the consensus-binding motif,
ATTAGCAT, the POUS subunit binds to GCAT and POUHD binds to ATTA without making
contact between subunits. Oct-4 was first detected in F9 embryonal carcinoma cells and named
NF-A3 (Lenardo et al., 1989). It was then independently cloned in P19 embryonal carcinoma
(Okamoto et al., 1990) and in the early developing mouse (Rosner et al., 1990) and named Oct-3
or in the pre-implantation mouse embryo and named Oct-4 (Scholer et al., 1990).
Mouse
POU domain, class 5, transcription factor 1 (Pou5f1) (GeneID: 18999) is located on
chromosome 17 in the mouse. Through five exons a 1346 nucleotide message (Genbank:
NM_013633) is transcribed resulting in a 352 amino acid protein. The POUS domain is found
between amino acids 131-205 and the homeodomain is located at position 224-282.
Human
The human POU class 5 homeobox 1 gene generates a transcript of 1417 nucleotides
(Genbank: NM_002701) from five exons. Within the 360 amino acids are the POU domain
(138-212) and the homeodomain (231-289).
Farm Animals
In the bovine, POU5F1 (GeneID: 282316) is located on chromosome 23 and encodes a
1615 base pair transcript (Genbank: NM_174580) which translates to a 360 amino acid protein.
It shares 90.6% and 81.7% overall identity with the human and mouse proteins respectively (van
Eijk et al., 1999). Porcine POU5F1 (GeneID: 100127461) is located on chromosome 7 and has a
coding sequence of 1083 base pairs (Genbank: NM_001113060) for a 360 amino acid. Identity
between the pig and bovine protein is 96.4%. The POUS region is between amino acids 138-212
and the POUHD lies between 231-289 for both the bovine and the pig.
4
Sox-2
SOX-2 belongs to a superfamily of DNA-proteins (SRY-related HMG box) including
SRY and its homologs. These proteins contain a single HMG box that binds the minor groove of
DNA in a highly sequence-specific manner (A/TCAAAG/C).
Mouse
The mouse Sox-2 gene (GeneID: 20674) produces a 2457 base pair transcript (Genbank:
NM_011443) that encodes a 319 amino acid protein. The HMG box is 81 amino acids (42-113).
Human
A 2518 base pair message (Genbank NM_003106) is transcribed by the Human SOX-2
gene (GeneID: 6657) resulting in a 317 amino acid protein.
Bovine
Bovine Sox-2 (GeneId: 784383) is 1477 nucleotides (Genbank NM_001105463)
resulting in a protein of 320 amino acids.
Gene Regulation
Regulation of Nanog
A composite Octamer/Sox binding site is found in the mouse Nanog promoter
approximately 180 nucleotides from the transcription start site (Kuroda et al., 2005; Rodda et al.,
2005). Using constructs from both the mouse and human promoter, mutations to either or both
elements dramatically reduced Nanog reporter expression in human and mouse ES cells (Kuroda
et al., 2005). Sox-2 and Oct-4 protein were also shown to bind to the Nanog promoter by
electrophoretic mobility shift assays and in vivo by chromatin immunoprecipitation assays
(Rodda et al., 2005). However, in Oct-4 deficient embryos Nanog expression is still observed
(Chambers et al., 2003). An unidentified factor, termed pluripotential cell-specific Sox element-
binding protein (PSBP), was reported in R1 mouse embryonic stem cells but not embryonic germ
or embryonal carcinoma cells (Kuroda et al., 2005). This finding could not be confirmed in a
different embryonic stem cell line (Rodda et al., 2005).
Nanog expression levels dropped by 15% when promoter constructs were shortened from
2342 base pairs to 332 base pairs demonstrating other elements upstream of the Oct/Sox element
5
are present that can regulate Nanog expression (Kuroda et al., 2005). Originally termed Genesis
because its expression was limited to pluripotent ES and EC cells (Sutton et al., 1996), FoxD3
belongs to the forkhead family of transcription factors and can up-regulate Nanog expression in
mouse ES and EC cells by binding to an ES cell specific enhancer E2-like element in the Nanog
promoter in vivo (Pan et al., 2006).
Pan et al., (2006) reported an Oct-4 dose-dependant effect on Nanog. At sub-steady
concentrations Oct-4 up-regulates Nanog however at higher concentrations Oct-4 may repress
Nanog. The tumor suppressor p53 has also been shown to down-regulate Nanog when
embryonic stem cells have experienced DNA damage (Lin et al., 2005) and a member of the
canonical Wnt signaling pathway, Tcf3, can also down-regulate Nanog expression (Pereira et al.,
2006).
Overexpression of Nanog increased the stability of undifferentiated cells by becoming
independent of LIF and are more resistant to differentiation procedures (Chambers et al., 2003).
Evidence suggests that Nanog can be a repressor of genes, especially those of endoderm lineage.
Differentiated Nanog null cells (-/-) expressed only endoderm markers such as gata4, gata6, tm,
and bmp2 (Mitsui et al., 2003) and when Nanog was down-regulated by RNA interference in
mouse embryonic stem cells, gata6, gata4, and laminin B1 were up-regulated (Hough et al.,
2006). Results differed in heterozygous Nanog (+/-) ES cells. With a feeder layer they can
remain undifferentiated but without out a feeder layer, the cells differentiated into endodermal,
mesodermal, and ectodermal lineages (Hatano et al., 2005).
Initially Nanog was hypothesized to be only a repressor of genes leading to
differentiation. However it has been shown the Nanog could be a potent activator of gene
transcription. The 10 pentapeptide repeat that begins with tryptophan and a second subdomain
located C-terminal to the repeat increased transcriptional activity of reporter constructs (Pan and
Pei, 2005). When the tryptophans in the first subdomain were substituted with alanines, activity
was abolished. More evidence suggests that Nanog, along with other transcription factors
implicated in pluripotency are all involved in extensive autoregulatory feedforward loops that
regulate their own expression as well as the expression of others. Nanog can activate expression
of Oct-4 (Pan et al., 2006) and Sall4 (Wu et al., 2006), a transcription factor that is expressed in
the inner cell mass of mouse embryos (Yoshikawa et al., 2006), embryonic carcinoma cells
(Kohlhase et al., 2002), and in the trophectoderm (Sakaki-Yumoto et al., 2006; Yoshikawa et al.,
6
2006). Another marker of pluripotent cells, Rex-1, is also up-regulated by Nanog in cooperation
with Sox-2 (Shi et al., 2006).
Regulation of Oct-4
Typically regulation of transcription is thought of as being under an off-on type of
control. To sustain self-renewal of mouse embryonic stem cells, the expression of Oct-4 must
fall within a critical level. Using an Oct-4 transgene under tetracycline control, Niwa and
coworkers (2000) were able to alter levels of Oct-4 expression. Relative to endogenous levels of
expression, a 1.5 fold decrease resulted in dedifferentiation into trophectoderm whereas a 1.5
fold increase resulted in differentiation into primitive endoderm and mesoderm. One way that
this steady-state regulation occurs is by negative feedback of Oct-4 on itself (Pan et al., 2006).
In comparing the bovine, mouse, and human Oct-4 promoters, van Eijk et al., (1999)
reported the presence of a highly conserved Sp 1 binding site and an overlapping hormone
responsive element. They also noted a Short Interspersed Nuclear Element (SINE) within the
bovine reporter was not found in the mouse.
Regulation of Sox-2
Wiebe and coworkers (2000) described a CCAAT box -60 base pairs from the
transcription start site that has a role in Sox-2 expression in undifferentiated F9 embryonal
carcinoma cells. However, it was still active after cell differentiation. An enhancer in the 3’-
flanking region of the Sox-2 gene has been described (Tomioka et al., 2002). Called Sox
regulatory region 2 (SRR2), it contains an Octamer/Sox-2 like recognition sequence that both
Oct-4/Sox-2 and Oct 6/ Sox-2 complexes can bind to and up-regulate expression. Zappone and
coworkers (2000) describes a regionally restricted enhancer upstream of the Sox-2 gene that is
active in mouse blastocysts but later becomes restricted to the developing telencephalon.
Gene Expression
Embryonic Development
Mouse
In the mouse, Nanog expression is first detected by northern blots in the compacted
morula and is confined solely to the inner cell mass and epiblast where it is down-regulated at
7
the time of implantation (Chambers et al., 2003; Mitsui et al., 2003). Nanog null embryos at 3.5
dpc appeares normal but by 5.5 dpc they are disorganized with no discernible epiblast or
extraembryonic ectoderm suggesting that Nanog is needed for maintenance of the epiblast
(Mitsui et al., 2003). Nanog expression is absent in mouse primordial germ cells that express
PGC7/Stella up to 7.5 dpc (Hatano et al., 2005; Yamaguchi et al., 2005) but by 7.75 dpc it is
expressed by primordial germ cells throughout migration and population of the early gonad until
female germ cells entered meiosis or the onset of mitotic arrest in male germ cells (Yamaguchi et
al., 2005).
Oct-4 is present within the mouse oocyte (Rosner et al., 1990) but the maternal message
is cleared by the 2-cell stage (Palmieri et al., 1994). Expression of Oct-4 occurs in the cells of
the morula, the inner cell mass of the blastocyst, and the primitive ectoderm of the egg cylinder
until the time of gastrulation (7.0 dpc) (Rosner et al., 1990). Rosner et al., (1990) also noted Oct-
4 message in the trophectoderm at 3.5 dpc while it was gone by 4.5 dpc, but this pattern has not
been observed by others (Palmieri et al., 1994). After gastrulation, Oct-4 was observed in
primordial germs migrating at 10.5 dpc and populating the genital ridges (Rosner et al., 1990).
Up-regulation of Oct-4 was observed in the early cells of primitive endoderm (Palmieri et al.,
1994). Thus a model has been proposed that Oct-4 can direct cell differentiation to
trophectoderm by down-regulation or to endoderm by up-regulation (Pesce and Scholer, 2001).
Other evidence for this model is provided by loss of the inner cell mass with only trophoblast
present in Oct-4 null embryos (Nichols et al., 1998), the up-regulation of trophectoderm markers
Cdx2, Hand1, and PL-1 in Oct4 silenced embryonic stem cells (Hough et al., 2006), and the up-
regulation of the endoderm marker gata4 in cells where Oct-4 was over expressed (Niwa et al.,
2000).
Using a reporter construct, Sox-2 expression is first detected in the morula and the inner
cell mass of the blastocyst and in later stages becomes confined to the neuroectoderm, the
extraembryonic ectoderm or chorion, and gut endoderm (Avilion et al., 2003). Sox-2 is required
for the maintenance of the epiblast and development of the extraembryonic ectoderm (Avilion et
al., 2003).
Farm Animal
The expression pattern of Oct-4, Sox-2, and Nanog is incompletely described in farm
animal species and appears to differ from that of the mouse where these markers are expressed in
8
only the undifferentiated cells of the mouse embryo. Oct-4 message was present in the inner cell
mass but not in the trophoblast of d-7 in vitro-derived bovine blastocysts (Kurosaka et al., 2004)
but was detected in the trophoblast of half of the bovine blastocysts derived by somatic cell
nuclear transfer (Wuensch et al., 2007). In pre-implanting bovine embryos, Oct-4 message was
present in both the embryonic and extraembryonic tissues but was detectable only in the
embryonic tissues of filamentous embryos (Degrelle et al., 2005). They also showed by in situ
hybridization, Oct-4 in the extraembryonic mesoderm of the elongating embryo. The Oct4
protein however, is present in both the inner cell mass and trophectoderm of bovine and porcine
blastocysts (Kirchhof et al., 2000; van Eijk et al., 1999) but becomes confined to the epiblast of
porcine and bovine embryos after hatching (Vejlsted et al., 2005; Vejlsted et al., 2006). By day
17 post-insemination, Oct-4 protein is generally cleared from the embryo except for the
presumptive primordial germ cells in the yolk sac endoderm and allantois (Vejlsted et al., 2006).
Nanog and Sox-2 expression in farm animals has only been reported in pre-implanting
bovine embryos, characterized as spherical, ovoid, and filamentous (Degrelle et al., 2005).
Using semi-quantitative PCR, these investigators found both Nanog and Sox-2 are expressed in
embryonic tissue at all stages and are up-regulated in the filamentous embryo. Sox-2 expression
is low in the extraembryonic tissues at spherical and ovoid stages and Nanog expression
increases in the extraembryonic tissues as development progresses.
9
Literature Cited
Avilion, A. A., S. K. Nicolis, L. H. Pevny, L. Perez, N. Vivian, and R. Lovell-Badge. 2003. Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev. 17:126-140.
Booth, H. A. and P. W. Holland. 2004. Eleven daughters of NANOG. Genomics 84:229-238.
Chambers, I., D. Colby, M. Robertson, J. Nichols, S. Lee, S. Tweedie, and A. Smith. 2003. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113:643-655.
Degrelle, S. A., E. Campion, C. Cabau, F. Piumi, P. Reinaud, C. Richard, J. P. Renard, and I. Hue. 2005. Molecular evidence for a critical period in mural trophoblast development in bovine blastocysts. Dev. Biol. 288:448-460.
Hatano, S. Y., M. Tada, H. Kimura, S. Yamaguchi, T. Kono, T. Nakano, H. Suemori, N. Nakatsuji, and T. Tada. 2005. Pluripotential competence of cells associated with Nanog activity. Mech. Dev. 122:67-79.
Herr, W. and M. A. Cleary. 1995. The POU domain: versatility in transcriptional regulation by a flexible two-in-one DNA-binding domain. Genes Dev. 9:1679-1693.
Herr, W., R. A. Sturm, R. G. Clerc, L. M. Corcoran, D. Baltimore, P. A. Sharp, H. A. Ingraham, M. G. Rosenfeld, M. Finney, G. Ruvkun, and . 1988. The POU domain: a large conserved region in the mammalian pit-1, oct-1, oct-2, and Caenorhabditis elegans unc-86 gene products. Genes Dev. 2:1513-1516.
Hough, S. R., I. Clements, P. J. Welch, and K. A. Wiederholt. 2006. Differentiation of mouse embryonic stem cells after RNA interference-mediated silencing of OCT4 and Nanog. Stem Cells 24:1467-1475.
Kirchhof, N., J. W. Carnwath, E. Lemme, K. Anastassiadis, H. Scholer, and H. Niemann. 2000. Expression pattern of Oct-4 in preimplantation embryos of different species. Biol. Reprod. 63:1698-1705.
Kohlhase, J., M. Heinrich, L. Schubert, M. Liebers, A. Kispert, F. Laccone, P. Turnpenny, R. M. Winter, and W. Reardon. 2002. Okihiro syndrome is caused by SALL4 mutations. Hum. Mol. Genet. 11:2979-2987.
Kuroda, T., M. Tada, H. Kubota, H. Kimura, S. Y. Hatano, H. Suemori, N. Nakatsuji, and T. Tada. 2005. Octamer and Sox elements are required for transcriptional cis regulation of Nanog gene expression. Mol. Cell Biol. 25:2475-2485.
10
Kurosaka, S., S. Eckardt, and K. J. McLaughlin. 2004. Pluripotent lineage definition in bovine embryos by Oct4 transcript localization. Biol. Reprod. 71:1578-1582.
Lenardo, M. J., L. Staudt, P. Robbins, A. Kuang, R. C. Mulligan, and D. Baltimore. 1989. Repression of the IgH enhancer in teratocarcinoma cells associated with a novel octamer factor. Science 243:544-546.
Lin, T., C. Chao, S. Saito, S. J. Mazur, M. E. Murphy, E. Appella, and Y. Xu. 2005. p53 induces differentiation of mouse embryonic stem cells by suppressing Nanog expression. Nat. Cell Biol. 7:165-171.
Mitsui, K., Y. Tokuzawa, H. Itoh, K. Segawa, M. Murakami, K. Takahashi, M. Maruyama, M. Maeda, and S. Yamanaka. 2003. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113:631-642.
Nichols, J., B. Zevnik, K. Anastassiadis, H. Niwa, D. Klewe-Nebenius, I. Chambers, H. Scholer, and A. Smith. 1998. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95:379-391.
Niwa, H., J. Miyazaki, and A. G. Smith. 2000. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat. Genet. 24:372-376.
Oh, J. H., H. J. Do, H. M. Yang, S. Y. Moon, K. Y. Cha, H. M. Chung, and J. H. Kim. 2005. Identification of a putative transactivation domain in human Nanog. Exp. Mol. Med. 37:250-254.
Okamoto, K., H. Okazawa, A. Okuda, M. Sakai, M. Muramatsu, and H. Hamada. 1990. A novel octamer binding transcription factor is differentially expressed in mouse embryonic cells. Cell 60:461-472.
Palmieri, S. L., W. Peter, H. Hess, and H. R. Scholer. 1994. Oct-4 transcription factor is differentially expressed in the mouse embryo during establishment of the first two extraembryonic cell lineages involved in implantation. Dev. Biol. 166:259-267.
Pan, G., J. Li, Y. Zhou, H. Zheng, and D. Pei. 2006. A negative feedback loop of transcription factors that controls stem cell pluripotency and self-renewal. FASEB J. 20:1730-1732.
Pan, G. and D. Pei. 2005. The stem cell pluripotency factor NANOG activates transcription with two unusually potent subdomains at its C terminus. J. Biol. Chem. 280:1401-1407.
Pan, G. J. and D. Q. Pei. 2003. Identification of two distinct transactivation domains in the pluripotency sustaining factor nanog. Cell Res. 13:499-502.
Pereira, L., F. Yi, and B. J. Merrill. 2006. Repression of Nanog gene transcription by Tcf3 limits embryonic stem cell self-renewal. Mol. Cell Biol. 26:7479-7491.
Pesce, M. and H. R. Scholer. 2001. Oct-4: gatekeeper in the beginnings of mammalian development. Stem Cells 19:271-278.
11
Rodda, D. J., J. L. Chew, L. H. Lim, Y. H. Loh, B. Wang, H. H. Ng, and P. Robson. 2005. Transcriptional regulation of nanog by OCT4 and SOX2. J. Biol. Chem. 280:24731-24737.
Rosner, M. H., M. A. Vigano, K. Ozato, P. M. Timmons, F. Poirier, P. W. Rigby, and L. M. Staudt. 1990. A POU-domain transcription factor in early stem cells and germ cells of the mammalian embryo. Nature 345:686-692.
Sakaki-Yumoto, M., C. Kobayashi, A. Sato, S. Fujimura, Y. Matsumoto, M. Takasato, T. Kodama, H. Aburatani, M. Asashima, N. Yoshida, and R. Nishinakamura. 2006. The murine homolog of SALL4, a causative gene in Okihiro syndrome, is essential for embryonic stem cell proliferation, and cooperates with Sall1 in anorectal, heart, brain and kidney development. Development 133:3005-3013.
Scholer, H. R., S. Ruppert, N. Suzuki, K. Chowdhury, and P. Gruss. 1990. New type of POU domain in germ line-specific protein Oct-4. Nature 344:435-439.
Shi, W., H. Wang, G. Pan, Y. Geng, Y. Guo, and D. Pei. 2006. Regulation of the pluripotency marker Rex-1 by Nanog and Sox2. J. Biol. Chem. 281:23319-23325.
Sutton, J., R. Costa, M. Klug, L. Field, D. Xu, D. A. Largaespada, C. F. Fletcher, N. A. Jenkins, N. G. Copeland, M. Klemsz, and R. Hromas. 1996. Genesis, a winged helix transcriptional repressor with expression restricted to embryonic stem cells. J. Biol. Chem. 271:23126-23133.
Tomioka, M., M. Nishimoto, S. Miyagi, T. Katayanagi, N. Fukui, H. Niwa, M. Muramatsu, and A. Okuda. 2002. Identification of Sox-2 regulatory region which is under the control of Oct-3/4-Sox-2 complex. Nucleic Acids Res. 30:3202-3213.
van Eijk, M. J., M. A. van Rooijen, S. Modina, L. Scesi, G. Folkers, H. T. van Tol, M. M. Bevers, S. R. Fisher, H. A. Lewin, D. Rakacolli, C. Galli, C. de Vaureix, A. O. Trounson, C. L. Mummery, and F. Gandolfi. 1999. Molecular cloning, genetic mapping, and developmental expression of bovine POU5F1. Biol. Reprod. 60:1093-1103.
Vejlsted, M., B. Avery, M. Schmidt, T. Greve, N. Alexopoulos, and P. Maddox-Hyttel. 2005. Ultrastructural and immunohistochemical characterization of the bovine epiblast. Biol. Reprod. 72:678-686.
Vejlsted, M., H. Offenberg, F. Thorup, and P. Maddox-Hyttel. 2006. Confinement and clearance of OCT4 in the porcine embryo at stereomicroscopically defined stages around gastrulation. Mol. Reprod. Dev. 73:709-718.
Wiebe, M. S., P. J. Wilder, D. Kelly, and A. Rizzino. 2000. Isolation, characterization, and differential expression of the murine Sox-2 promoter. Gene 246:383-393.
Wu, Q., X. Chen, J. Zhang, Y. H. Loh, T. Y. Low, W. Zhang, W. Zhang, S. K. Sze, B. Lim, and H. H. Ng. 2006. Sall4 interacts with Nanog and co-occupies Nanog genomic sites in embryonic stem cells. J. Biol. Chem. 281:24090-24094.
12
Wuensch, A., F. A. Habermann, S. Kurosaka, R. Klose, V. Zakhartchenko, H. D. Reichenbach, F. Sinowatz, K. J. McLaughlin, and E. Wolf. 2007. Quantitative monitoring of pluripotency gene activation after somatic cloning in cattle. Biol. Reprod. 76:983-991.
Yamaguchi, S., H. Kimura, M. Tada, N. Nakatsuji, and T. Tada. 2005. Nanog expression in mouse germ cell development. Gene Expr. Patterns. 5:639-646.
Yoshikawa, T., Y. Piao, J. Zhong, R. Matoba, M. G. Carter, Y. Wang, I. Goldberg, and M. S. Ko. 2006. High-throughput screen for genes predominantly expressed in the ICM of mouse blastocysts by whole mount in situ hybridization. Gene Expr. Patterns. 6:213-224.
Zappone, M. V., R. Galli, R. Catena, N. Meani, S. De Biasi, E. Mattei, C. Tiveron, A. L. Vescovi, R. Lovell-Badge, S. Ottolenghi, and S. K. Nicolis. 2000. Sox2 regulatory sequences direct expression of a (beta)-geo transgene to telencephalic neural stem cells and precursors of the mouse embryo, revealing regionalization of gene expression in CNS stem cells. Development 127:2367-2382.
13
CHAPTER 2 - Cloning and Expression of Pluripotent Factors
Around the Time of Gastrulation in the Porcine Conceptus
Introduction After fertilization, the embryo will undergo a series of cleavage divisions resulting in the
morula. The first appearance of cell differentiation occurs at blastocyst formation as the outer
cells of the morula differentiate into trophoblast. The inner cell mass is comprised of pluripotent
cells that will give rise to all three germ layers of developing embryo in the process of
gastrulation. The transcription factors important in maintaining pluripotency in the inner cell
mass and the ensuing epiblast include Nanog, Sox-2, and Oct-4. Nanog is a homeobox
transcription factor that is first observed in the morula and becomes confined to the inner cell
mass and epiblast in the mouse (Chambers et al., 2003; Mitsui et al., 2003). In the mouse, the
loss of Nanog results in disorganized tissue with no discernable epiblast at day 5.5 post-coitum
(Mitsui et al., 2003). Oct-4 is also expressed early in mouse development (Palmieri et al., 1994;
Rosner et al., 1990) and is required for a pluripotent inner cell mass (Nichols et al., 1998).
Consistent with Nanog and Oct-4, Sox-2 is also expressed in the inner cell mass and is required
to maintain the epiblast (Avilion et al., 2003) but unlike the other factors it continues to be
expressed in a differentiated tissue, the developing nervous system (Uchikawa et al., 1999;
Uwanogho et al., 1995; Wood and Episkopou, 1999). In embryonic stem cells, these factors
suppress differentiation and promote self-renewal by forming an autoregulatory and feedforward
network (Boyer et al., 2005; Loh et al., 2006; Wang et al., 2006).
The expression pattern of these markers in farm animal species is not well characterized
and may differ from that of the mouse. Oct-4 is observed in the trophoblast of porcine and
bovine blastocysts (Kirchhof et al., 2000; van Eijk et al., 1999) and expression of these markers
is observed in the extraembryonic tissues of the bovine prior to implantation (Degrelle et al.,
2005). Therefore, we have partially cloned the porcine Oct-4, Nanog, and Sox-2 transcripts and
characterize their expression in day 10, 12, 15, and 17 embryonic and extraembryonic tissues as
well as endometrium, myometrium, placenta, and fetal liver at day 40 of pregnancy.
14
Material and Methods
Tissue Collection
Embryos were flushed from sows under general anesthesia 10 (n=3), 12 (n=4), 15 (n=5),
and 17 (n=3) days post-insemination by exposing the reproductive tract through a mid-ventral
incision. The uterine horn was clamped near the uterine body and 50 ml of 37oC DMEM was
injected at the utero-tubal junction. Medium was flushed through a small incision above the
clamp into 100 mM Petri dishes. Day-10 and -12 embryos were processed as whole conceptuses
(embryonic and extraembryonic tissues). Day-15 and -17 embryonic tissue (embryonic disk)
was separated by closely trimming the adjacent extraembryonic tissues (proximal
extraembryonic) with a scalpel under a stereo-microscope (5 to 50X). Additional
extraembryonic tissue was collected after removal of the embryonic disks (distal
extraembryonic). In addition, d-17 allantois and whole embryos (embryonic plus
extraembryonic tissue still attached) were isolated. Day-40 endometrium, myometrium,
placenta, and fetal liver were collected as previously described (Brown et al., 2007).
RNA and Protein Isolation
Total RNA was isolated from porcine embryonic and extraembryonic tissue at day 10,
12, 15, and 17 using the RNeasy Mini or RNeasy Micro Kits (Qiagen; Valencia, CA) according
to manufacturer’s instructions. Tissue was disrupted by a buffer containing guanidine
thiocyanate and β-mercaptoethonal and homogenized by passing the lysate through a
QIAshredder spin column (Qiagen). RNA was DNase treated by the on-column DNA digestion
procedure in the kit instructions. Total RNA was quantified using the NanoDrop ND-1000
Spectrophotometer (NanoDrop Technologies; Wilmington, DE) and run on a 1% agarose-
formaldehyde gel to assess quality. Protein from porcine subdermal skin cells, D3 mouse
embryonic stem cells, d-15 extraembryonic tissue, and d-15 and -17 conceptus (embryonic and
extraembryonic) were isolated by incubating cell cultures or tissues with M-PER Mammalian
Protein Extraction Reagent (Pierce; Rockford, IL) for ~10 minutes and then samples were
centrifuged at 13,200 g for 10 minutes. Supernatant was transferred to clean tube and stored at -
70oC.
15
Gene Cloning
Full-length, RNA ligase-mediated (RLM) rapid amplification of the 5’ end of Nanog was
performed with the FirstChoice RLM-RACE Kit (Ambion; Austin, TX) following manufacture's
instructions. Briefly, total RNA was incubated at 37oC for 1 h with calf alkaline phosphatase to
remove 5’ phosphates from truncated mRNA and non-mRNA, and then treated with tobacco acid
pyrophosphatase at 37oC for 1 h to remove the cap structure from full-length mRNA. An RNA
oligonucleotide was then ligated to decapped RNA using T4 RNA ligase. The RT reaction for
the 5’ end was performed using the M-MLV reverse transcriptase and random decamer primers.
The 3’ ends of all transcripts were obtained using the GeneRacer Kit (Invitrogen, Carlsbad CA).
First-strand cDNA was synthesized using SuperScript III reverse transcriptase and an Oligo dT
primer creating a known priming site at the 3’ end. The promoter sequence was cloned by PCR
from DNA isolated from porcine whole blood.
Reaction conditions for the 5’ outer (25 ul total) and inner (50 ul total) reaction were as
follows; 0.5 ul first-strand cDNA (outer) or 2.0 ul outer PCR reaction (inner), 1X Amplitaq
buffer with MgCl2 [1.5mM], 5’ RACE gene-specific outer primer and Nanog outer primer
(outer) or 5’ RACE gene-specific inner primer and Nanog inner primer (inner) [0.4 uM each],
dNTPs [200 uM each], and 1.5 U of Amplitaq polymerase. Cycling conditions for both the
initial and nested PCR of the 5’ end were as follows: 3 minutes at 94oC followed by 35 cycles at
94oC for 30 sec, 60oC for 30 sec, and 72oC for 30 sec concluding with 72oC for 7 min.
5’ RACE
Primer Sequence (5’ – 3’) Predicted Annealing Temp (ºC)
Nanog outer GTCTGGTTGCTCCAGGTTG 55
Nanog inner AGAAGCGTTCACCAGGCAT 57
3’ RACE
β-actin outer ACCACTGGCATTGTCATGGACTCT 63
Nanog outer ATCCAGCTTGTCCCCAAAG 56
Nanog inner AGCCTCCAGCAGATGCAAG 58
16
Oct-4 outer AGGTGTTCAGCCAAACGACC 58
Oct-4 inner GCTGCAGAAGTGGGTGGAGGAAG 65
Sox-2 outer ACAACTCGGAGATCAGCAAGCG 62
Sox-2 inner GCCTGGGCGCCGAGTGGA 69
Nanog Promoter
PromoterFwd TGTGACCTTAGAGTGAACCAAAGA 57
PromoterRev TGACATCTGCAAGGAGGCATA 58
Reaction conditions for the 3’ end were similar to the 5’ end conditions except
primer concentrations in the outer reaction were 0.6 uM for the GeneRacer 3’ primer and 0.2 uM
for the gene-specific outer primer. Cycling conditions for the outer reaction were 94oC for 2
minutes, 5 cycles at 94oC for 30 sec and 70oC for 2 minutes, 5 cycles at 94oC for 30 sec and 68oC
for 2 minutes and then 25 cycles at 94oC for 30 sec, 58oC for 30 sec, and 70oC for 2 minutes
concluding with 70oC for 10 min. The 3’ nested reaction consisted of 2 minutes at 94oC
followed by 25 cycles at 94oC for 30 sec, 64oC for 30 sec, and 68oC for 2 min concluding with
68oC for 10 min. Cloning of the PCR products was performed by using the TOPO TA Cloning
kit (Invitrogen; Carlsbad, CA). Plasmid DNA was isolated using Miniprep kit (Qiagen) and
sequenced using M13 forward and reverse priming sites by DNA Sequencing Core Facility
(University of Arkansas for Medical Sciences; Little Rock, AR).
Real-time PCR
One microgram of total RNA was reverse transcribed in a 50 ul reaction using TaqMan
Reverse Transcription Reagents (Applied Biosystems; Foster City, CA). The reaction
components included 5.0 ul RT buffer [1X], 11.0 ul MgCl2 [5.5mM], 10.0 ul dNTPs [500 µM
each], 2.5 ul random hexamers [2.5 µM], 1.0 ul inhibitor [20 U], and 3.2 ul Multiscribe reverse
transciptase [160 U]. Cycling conditions were 25.0ºC for 10 minutes, 37.0ºC for 60 minutes and
95ºC for 5 minutes. Reactions using less the one microgram were adjusted proportionally.
17
Primer and probe sequences were obtained using Primer Express (Applied Biosystems)
using the porcine Nanog and Sox-2 sequences and bovine Oct-4 sequence. Primers were tested
before using the TaqMan probe in real-time PCR assays. cDNA was serially diluted 10 fold
from 1:1 to 1:10000 and real-time PCR was performed at each dilution using 1X SYBR Green
PCR Master Mix (Applied Biosystems) and 300 uM forward and reverse primers in a 20ul
reaction. Melting curves were used to confirm the synthesis of a single PCR product. Threshold
values for each dilution point were plotted to calculate the slope. Primer efficiency was
estimated by efficiency = 10(-1/m)-1where m = slope. Primer efficiencies ranged from ~90-110%.
For real-time PCR reactions, 1 ul of cDNA was added to the following; TaqMan
Universal PCR Master Mix [1X], fwd primer [900 nM], reverse primer [900 nM], TaqMan
TAMRA probe [250 nM] in a 20 ul reaction. 18s ribosomal RNA was used as the normalization
control using TaqMan Ribosomal RNA Control Reagents (Applied Biosystems). Cycling
conditions were 50.0ºC for 2 min, 95.0ºC for 10 minutes, and 40 cycles of 95.0ºC for 15 sec and
60ºC for 1 min. The GLM procedure of SAS (SAS Institute Inc.; Cary, NC) was used to analyze
threshold values adjusted for 18s with tissue as the fixed effect.
Primer Sequence (5’ – 3’) Positiona Predicted Annealing Temp (ºC)
Predicted Size (bp)
Nanog fwd CCCGGGCTTCTATTCCTACCA 715 61 68
Nanog rev TACCCCACACGGGCAGGTT 782 62
Nanog Probe CAAGGATGCCTGGTGAACGCTTCTG 737 68
Oct-4 fwd GCAAGGCAGAGACCCTTGTG 31 59 68
Oct-4 rev GCCTCTCACTCGGTTCTCGAT 98 59
Oct-4 probe AGGCCCGAAAGAGAAAGCGGACG 52 69
Sox-2 fwd TTCCATGGGCTCAGTGGTCAA 493 62
Sox-2 rev TGGAGTGGGAAGAAGAGGTAAC 563 56
Sox-2 probe TCCGAGGCGAGCTCCAGCCC 515 70 71
aPosition based on clone sequences. See Figures 1, 5, and 6
18
Northern Blotting
10-20 ug of total RNA was separated in a 1% denaturing agarose/formaldehyde gel for 4
hours at 50 volts. Two micrograms of RNA Millennium Markers (Ambion) was used as RNA
standards. The gel was rinsed 4X in distilled H20 before alkaline transfer to Nytran SuPer
Charge nylon membrane using the Turboblotter Rapid Downward Transfer System and blotter
pack following manufacture’s instructions (Schleicher & Schuell, Keene, N.H.). Transfer buffer
consisted of 3 M NaCl and 8 mM NaOH. Transfer was allowed to occur for 4 hours and then
membrane was neutralized for 5 min. in neutralization buffer (1 M phosphate, pH 6.8).
Membranes were wrapped in clear plastic wrap and exposed to UV light for 30 seconds to
covalently bind RNA to membrane.
Probe labeling and detection of RNA was done through the Amersham Gene Images
AlkPhos Direct Labelling and Detection System (GE Healthcare, Piscataway, NJ) according to
manufacturers instructions. Probes were synthesized by PCR using plasmid DNA generated
from the cloning experiments as the template. Bands were cut from the gel and isolated from the
agarose gel by the MinElute Gel Extraction Kit (Qiagen). Primer sequences were as follows:
Primer Sequence (5’ – 3’) Predicted Annealing Temp (ºC)
Predicted Size (bp)
Nanog fwd TCCAGCTTGTCCCCAAAGC 59 556
Nanog rev AGAAGCGTTCACCAGGCAT 57
Oct-4 fwd GCTGCAGAAGTGGGTGGAGGAAG 65 414
Oct-4 rev TCAGGGAAAGGCACCGAGGAGTA 64
Sox-2 fwd GCCTGGGCGCCGAGTGGA 69 563
Sox-2 rev TGGAGTGGGAGGAAGAGGTAAC 69
β-actin fwd ACCACTGGCATTGTCATGGACTCT 63 545
β-actin rev ATCTTGATCTTCATGGTGCTGGGC 64
19
To label probes, DNA (10 ng/uL) was first denatured by placing in a vigorously boiling
water bath for 5 minutes and then cooled 5 minutes on ice. Reaction buffer, labeling reagent,
and cross-linker working solution were mixed with the DNA and incubated at 37ºC for 30
minutes. Blots were placed in pre-warmed hybridization buffer containing NaCl [0.5 M] and
blocking reagent [4% w/v] for 15 minutes at 55ºC. Labeled probe (200 ng total) was added at 20
ml of buffer and left to hybridize overnight. Blots were washed 2X in a primary washing buffer
(2M Urea, SDS (0.1% w/v), 50 mM NaH2PO4 (pH 7.0) 150 mM NaCl, 1 mM MgCl2, and
blocking reagent (0.2% w/v)) at 55ºC for 10 minutes. Blots were then washed 2X in a secondary
wash buffer (in mM; 50 Tris, 100 NaCl, and 2 MgCl2) at room temperature for 5 minutes. Two
mL of CDP-Star detection reagent was placed on the blot for 5 minutes and excess was drained
before blot was wrapped in clear plastic wrap. Blots were exposed to Classic Blue
Autoradiography Film BX (MIDSCI, St. Louis, MO) initially for 15 min-1 hour and then a
second film was exposed overnight. Blots were stripped in 0.5% (w/v) SDS at 60ºC for 60
minutes and rinsed for 5 minutes in 100 mM Tris (pH 8.0) at room temperature.
Western Blotting
Approximately 20 ug of protein in Laemmli Sample buffer was loaded onto a 12% Tris-
HCl pre-cast gel (Bio-Rad) and run at 200V for 45 minutes. Prestained kaleidoscope and
biotinylated SDS-PAGE broad range were included as standards. Protein was transferred to
Immun-Blot PVDF membrane in a 1x Tris/Glycine buffer (25 mM Tris, 192 mM Glycine, 20%
Methanol (v/v), pH 8.3) for 1 hour at 100 volts. The Vectastain ABC Elite kit (Vector
Laboratories, Inc., Burlingame, CA) was used for detection of protein following manufacturer’s
instructions. Briefly, membranes were blocked for 30 minutes in a 1x casein solution which was
used in all remaining steps and washes. Membranes were incubated with primary antibody for
30-60 minutes. Primary antibodies and dilutions were as follows: rabbit anti-Nanog (Chemicon
International; Temecula, CA), goat anti-Oct3/4 (Santa Cruz Biotechnology; Santa Cruz, CA),
and goat anti-Sox-2 (R&D Systems, Inc; Minneapolis, MN) at 1:2500, 1:200, and 1:1000
respectively. After 3-4 washes, membranes were incubated with biotinylated secondary antibody
(1:200) for 30 minutes with gentle agitation, followed by incubation in Vectastain ABC reagent
for 30 minutes. The TMB Substrate Kit for Peroxidase (Vector Laboratories, Inc) was used for
staining.
20
Results
Cloning
A putative full length cDNA transcript of Nanog was synthesized comprised of 1181 bp
which encodes a protein of 304 amino acids (see Figure 1). The homeodomain is comprised of
58 amino acids (position 97-154) and c-terminal to the homeodomain is a tryptophan at every
fifth position repeated nine times. Comparison of the amino acid sequence with sequences of
other species (see Figure 2) revealed 75% identity with the human (Genbank NP_079141), 81%
with the bovine (Genbank NP_001020515), 83% with the caprine (Genbank AY786437), and <
60% in the mouse (Genbank NP_082292). Similarity within the homeodomain is high in all
species including 89% within the mouse, 98% in the bovine and caprine, and 94% with the
human protein
A 769 bp product was cloned through PCR which included 453 bp upstream of the
transcription start site and 316 bp of coding sequence (see Figure 3). The Octamer/Sox element
was identified at position -149 relative to the start site. The Octamer sequence (5’-CTTTGCAT-
3’) differs slightly from the consensus sequence by either a T deletion or C addition at the first
position. The Sox sequence is conserved. A putative binding site for FoxD3 is also found at
position -259. Comparison of the non-coding region with the homologous regions of the bovine
and human resulted in 70 and 71% identity in 419 and 328 bp respectively. Comparing those
sequences with the mouse resulted in ~75% identity but only in a 94 bp region (see Figure 4).
The Oct-4 sequence included 452 bp of coding sequence which resulted in 149 amino
acids of the protein and included the 57 amino acid POU homeodomain (see Figure 5). Overall
sequence similarity with the bovine, human, and mouse Oct-4 proteins were 96, 94, and 86%
respectively and identity within the homeodomain was 100, 96, and 88% respectively. Porcine
Oct-4 is similar to mouse Oct-4 in that 13 of the 71 amino acids C-terminal to the homeodomain
are prolines (Okamoto et al., 1990). Results when blasted against the porcine genome resulted in
2 matches on chromosome 7. The first was 164 base pairs with 100% identity to the genomic
sequence. The second match was separated by 98 base pairs and resulted in a 99.6% match. We
hypothesize that this corresponds to exon 4 and 5. A thymidine at base pair 87 was changed in
our cloned sequence to a cytosine based on comparison with the genomic sequence. This
resulted in a stop codon being translated to an arginine.
21
Two products differing by 57 bp in length at the 3’ end were cloned for Sox-2.
Combining the longer sequence with an earlier cloned sequence (Genbank: DQ159208) an 860
bp product was generated. The coding sequence is 755 bp in length and results in 250 amino
acids (see Figure 6). This includes a partial sequence of the HMG binding domain. This protein
is highly conserved; identity between mouse, human, and bovine protein is greater than 98%.
Real-time PCR
Expression of Nanog, Oct-4, and Sox-2 were measured in d-10 and -12 whole conceptus,
embryonic and extraembryonic tissues at d-15 and -17, d-40 fetal liver, and placental,
endometrial, and myometrial tissues recovered from d-40 of pregnancy. Adjusted threshold
means and standard errors are reported in Table 1.
Expression of Nanog was lowest for all extraembryonic tissues at day 15 and 17 (see
Figure 7). Nanog expression levels were similar in the allantois, d-10 and -12 conceptus, and d-
40 endometrium. Expression increased 1.5 fold in the d-15 and -17 disk, however this was not
significant. The highest expression of Nanog was surprisingly observed in the d-40 tissues fetal
liver, placenta, and myometrium which were 27, 39, and 72 fold higher than extraembryonic
tissue expression respectively.
Oct-4 expression (see Figure 8) was low in all d-40 tissues except fetal liver where
expression was approximately 26 fold higher than the other d-40 tissues. Expression was highest
in d-10 and -12 conceptuses, and d-15 disk but decreased 3.5 fold by d-17. Higher expression
(4-5 fold) was also observed in the proximal extraembryonic tissue compared to the distal
extraembryonic tissue but may also be declining between day 15 and day 17 (1.5 fold).
Expression in the allantois was 10 fold higher than compared to myometrium
Sox-2 expression (Figure 9) was significantly up-regulated by 45 fold in the d-15 disk
and 80 fold in the d-17 disk when compared to the d-12 conceptus. Expression in the fetal liver
was also high; 70 fold higher when compared to myometrium and endometrium expression
which showed the lowest expression. Allantois also had higher levels of Oct-4 expression (14
fold) when compared to the endometrium.
Northern Blots
Total RNA (10 ug) from d-12 conceptus, and d-15 disk, distal and proximal
extraembryonic tissues were blotted and probed for Sox-2. A band of approximately 2300 base
22
pairs in length was detected for embryonic disk (see Figure 10). The membrane was stripped
and reprobed for Oct-4 but no bands were detected. Tissues having the highest Nanog
expression and 20 ug of total RNA, from d-17 disk, allantois, and d-12 conceptus were used to
generate a new blot for Nanog. Even after ~18 hours exposure no bands could be visualized.
Both blots were then probed for β-actin and bands of ~1750 and 1550 bp were observed (see
Figure 11). The smaller bands were observed in the d-15 extraembryonic tissues and are likely a
transcript variant. According to the description of Human β-actin (GeneID 60), the gene encodes
6 different highly conserved proteins. Based on these results we conclude that our current
detection protocol is not sensitive enough to detect Oct-4 and Nanog.
Western Blots
Nanog protein was detected in porcine fibroblasts, mouse D3 cells, and d-15 conceptus
and extraembryonic tissue as a 50 kDa band (see Figure 12). A second band of 23 kDa was
observed in the porcine samples and a third band was observed at 63 kDa in the d-15 samples.
Discussion Sequencing of the porcine genes resulted in the complete coding sequence for Nanog and
partial sequences for Sox-2 and Oct-4. Nanog appears to be the least conserved when compared
to other species including the bovine, human, and mouse as overall identity with the porcine
protein ranges from 83% to < 60%. The low identity with the mouse is similar to the less than
60% identity between human and mouse (Chambers et al., 2003; Hart et al., 2004). However the
identity within the homeobox is high (> 89%) and the tryptophan repeat motif of five amino
acids (WXXXX) is conserved. The tryptophan repeats nine times as it does in the human but
maintains the tryptophans in each repeat whereas in the human a tryptophan is substituted with a
glutamine in the forth repeat. The repeat forms a subdomain that has transcriptional activation
properties that become lost when alanines are substituted for the tryptophans (Pan and Pei,
2005). The Nanog nucleotide sequence was blasted in the porcine genome and resulted in one
match on chromosome 1. We hypothesize that this may be a pseudogene because porcine Nanog
has been mapped to chromosome 5 (Yang et al., 2004) and that the matched sequence was
intronless, similar to the mouse pseudogene described by (Hatano et al., 2005). The porcine
chromosome 5 sequence has not yet been made available. The 50 kDa band for Nanog was larger
than the expected (37 kDa) but is consistent with other results for porcine Nanog including the
23
23 kDa band (Kei Miyamoto; Kyoto University, Japan; personal communication). Smaller bands
are seen in mouse Nanog (Hatano et al., 2005; Wu et al., 2006) and it is hypothesized that Nanog
is easily degraded.
Conserved within the porcine Nanog promoter are the Octamer/Sox element and a
putative FoxD3 binding site. The element is invariant among 5 species (Rodda et al., 2005) and
can up-regulate Nanog expression by Oct-4 and Sox-2 binding (Kuroda et al., 2005; Rodda et al.,
2005). The potential effect of a single base change observed in the porcine octamer sequence is
unknown but could be addressed by targeted mutagenesis.
Of the three transcription factors, Sox-2 expression in the pig is the most consistent with
the expression pattern seen in mouse development. Sox-2 is necessary for the maintenance of
the epiblast (Avilion et al., 2003) and is expressed during neural development in the embryonic
disk (Uchikawa et al., 1999; Uwanogho et al., 1995; Wood and Episkopou, 1999). The up-
regulation in the d-15 disk is concurrent with the formation of the neural tube in the pig (Vejlsted
et al., 2006) and similar up-regulation is observed in the elongated bovine embryo (Degrelle et
al., 2005). The high expression of Oct-4 in d-10 and -12 conceptuses and d-15 embryonic disk is
consistent with its role in maintaining pluripotency of the inner cell mass (Nichols et al., 1998)
but down-regulation at d-17 suggests that those cells are undergoing differentiation and that
gastrulation is occurring. The presence of Oct-4 message and protein in the extraembryonic
tissues of farm animals (Degrelle et al., 2005; Kirchhof et al., 2000; van Eijk et al., 1999)
remains to be clarified. The greater expression seen in the proximal extraembryonic tissue as
opposed to the distal extraembryonic tissue may be due to a population of trophoblast stem cells
that delays commitment to differentiate until elongation (Degrelle et al., 2005) and promotes
trophoblast proliferation through Oct-4-directed secretion of FGF4 (Nichols et al., 1998; Tanaka
et al., 1998).
The expression of Nanog early in development is also consistent with maintaining
pluripotent stem cells in the mouse epiblast (Chambers et al., 2003; Mitsui et al., 2003) but the
dramatic up-regulation in d-40 tissues was unexpected. It may be relevant that Nanog expression
in porcine umbilical cord matrix cells was similar to d-15 embryonic disk while Oct-4 and Sox-2
expression were reduced (Carlin et al., 2006). Nanog is thought to be a repressor of endoderm
(Hough et al., 2006; Mitsui et al., 2003) and mesoderm differentiation (Suzuki et al., 2006) and
an activator of pluripotent markers such as Sall4 (Wu et al., 2006), Rex1 (Shi et al., 2006), and
24
Oct-4 (Pan et al., 2006). However, in a pattern like Sox-2, Nanog may continue to be expressed
in a differentiated tissues and this warrants further study to determine it’s role and how it is being
regulated.
Relatively high expression of all three transcription factors in the d-40 liver suggests a
population of more primitive type cells. Progenitors of red blood cells from the yolk sac and
hematopoietic stem cells from the embryo populate the fetal liver where blood cell formation
occurs until near the end of gestation when it will moves to it’s final location in the bone marrow
(McGrath and Palis, 2008). Fetal tissues may have some expression of these factors based on the
number of reports of multipotent progenitor cells in adult tissues. From human adult liver, Oct-4
and Nanog expressing multipotent adult stem cells have been isolated (Beltrami et al., 2007).
Oct-4 has been reported in many adult tissues (Cervello et al., 2007; Matthai et al., 2006; Tai et
al., 2005) as well a Nanog (Hart et al., 2004). Stem cells in adult tissues serve to replace dying
cells or regenerate damaged tissue and these cells may have a role in tissue generation in the
developing embryo.
Nanog regulation has been characterized in mouse pluripotent stem cells (Kuroda et al.,
2005; Pan et al., 2006; Rodda et al., 2005; Wu et al., 2006) but to better understand Nanog
expression in tissues such as d-40 myometrium or placenta, we plan to further characterize
regulatory elements and transcription factors involved in Nanog expression using porcine
umbilical cord matrix cells. This would be the first report describing Nanog regulation in cells
other than the mouse from a non-embryonic source. The long-term goal is to create reporter
constructs that can be tested in vivo using embryos generated through somatic cell nuclear
transfer.
25
Literature Cited
Avilion, A. A., S. K. Nicolis, L. H. Pevny, L. Perez, N. Vivian, and R. Lovell-Badge. 2003. Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev. 17:126-140.
Beltrami, A. P., D. Cesselli, N. Bergamin, P. Marcon, S. Rigo, E. Puppato, F. D'Aurizio, R. Verardo, S. Piazza, A. Pignatelli, A. Poz, U. Baccarani, D. Damiani, R. Fanin, L. Mariuzzi, N. Finato, P. Masolini, S. Burelli, O. Belluzzi, C. Schneider, and C. A. Beltrami. 2007. Multipotent cells can be generated in vitro from several adult human organs (heart, liver, and bone marrow). Blood 110:3438-3446.
Boyer, L. A., T. I. Lee, M. F. Cole, S. E. Johnstone, S. S. Levine, J. P. Zucker, M. G. Guenther, R. M. Kumar, H. L. Murray, R. G. Jenner, D. K. Gifford, D. A. Melton, R. Jaenisch, and R. A. Young. 2005. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122:947-956.
Brown, K. R., R. D. Goodband, M. D. Tokach, S. S. Dritz, J. L. Nelssen, J. E. Minton, J. J. Higgins, J. C. Woodworth, and B. J. Johnson. 2007. Growth characteristics, blood metabolites, and insulin-like growth factor system components in maternal tissues of gilts fed L-carnitine through day seventy of gestation. J. Anim Sci. 85:1687-1694.
Carlin, R., D. Davis, M. Weiss, B. Schultz, and D. Troyer. 2006. Expression of early transcription factors Oct4, Sox2 and Nanog by porcine umbilical cord (PUC) matrix cells. Reprod. Biol. Endocrinol. 4:8.
Cervello, I., J. A. Martinez-Conejero, J. A. Horcajadas, A. Pellicer, and C. Simon. 2007. Identification, characterization and co-localization of label-retaining cell population in mouse endometrium with typical undifferentiated markers. Hum. Reprod. 22:45-51.
Chambers, I., D. Colby, M. Robertson, J. Nichols, S. Lee, S. Tweedie, and A. Smith. 2003. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113:643-655.
Degrelle, S. A., E. Campion, C. Cabau, F. Piumi, P. Reinaud, C. Richard, J. P. Renard, and I. Hue. 2005. Molecular evidence for a critical period in mural trophoblast development in bovine blastocysts. Dev. Biol. 288:448-460.
Gupta, S., C. Verfaillie, D. Chmielewski, S. Kren, K. Eidman, J. Connaire, Y. Heremans, T. Lund, M. Blackstad, Y. Jiang, A. Luttun, and M. E. Rosenberg. 2006. Isolation and characterization of kidney-derived stem cells. J. Am. Soc. Nephrol. 17:3028-3040.
Hart, A. H., L. Hartley, M. Ibrahim, and L. Robb. 2004. Identification, cloning and expression analysis of the pluripotency promoting Nanog genes in mouse and human. Dev. Dyn. 230:187-198.
26
Hatano, S. Y., M. Tada, H. Kimura, S. Yamaguchi, T. Kono, T. Nakano, H. Suemori, N. Nakatsuji, and T. Tada. 2005. Pluripotential competence of cells associated with Nanog activity. Mech. Dev. 122:67-79.
Hough, S. R., I. Clements, P. J. Welch, and K. A. Wiederholt. 2006. Differentiation of mouse embryonic stem cells after RNA interference-mediated silencing of OCT4 and Nanog. Stem Cells 24:1467-1475.
Kirchhof, N., J. W. Carnwath, E. Lemme, K. Anastassiadis, H. Scholer, and H. Niemann. 2000. Expression pattern of Oct-4 in preimplantation embryos of different species. Biol. Reprod. 63:1698-1705.
Kuroda, T., M. Tada, H. Kubota, H. Kimura, S. Y. Hatano, H. Suemori, N. Nakatsuji, and T. Tada. 2005. Octamer and Sox elements are required for transcriptional cis regulation of Nanog gene expression. Mol. Cell Biol. 25:2475-2485.
Loh, Y. H., Q. Wu, J. L. Chew, V. B. Vega, W. Zhang, X. Chen, G. Bourque, J. George, B. Leong, J. Liu, K. Y. Wong, K. W. Sung, C. W. Lee, X. D. Zhao, K. P. Chiu, L. Lipovich, V. A. Kuznetsov, P. Robson, L. W. Stanton, C. L. Wei, Y. Ruan, B. Lim, and H. H. Ng. 2006. The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat. Genet. 38:431-440.
Matthai, C., R. Horvat, M. Noe, F. Nagele, A. Radjabi, M. van Trotsenburg, J. Huber, and A. Kolbus. 2006. Oct-4 expression in human endometrium. Mol. Hum. Reprod. 12:7-10.
McGrath, K. and J. Palis. 2008. Chapter 1 ontogeny of erythropoiesis in the Mammalian embryo. Curr. Top. Dev. Biol. 82:1-22.
Mitsui, K., Y. Tokuzawa, H. Itoh, K. Segawa, M. Murakami, K. Takahashi, M. Maruyama, M. Maeda, and S. Yamanaka. 2003. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113:631-642.
Nichols, J., B. Zevnik, K. Anastassiadis, H. Niwa, D. Klewe-Nebenius, I. Chambers, H. Scholer, and A. Smith. 1998. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95:379-391.
Okamoto, K., H. Okazawa, A. Okuda, M. Sakai, M. Muramatsu, and H. Hamada. 1990. A novel octamer binding transcription factor is differentially expressed in mouse embryonic cells. Cell 60:461-472.
Palmieri, S. L., W. Peter, H. Hess, and H. R. Scholer. 1994. Oct-4 transcription factor is differentially expressed in the mouse embryo during establishment of the first two extraembryonic cell lineages involved in implantation. Dev. Biol. 166:259-267.
Pan, G., J. Li, Y. Zhou, H. Zheng, and D. Pei. 2006. A negative feedback loop of transcription factors that controls stem cell pluripotency and self-renewal. FASEB J. 20:1730-1732.
27
Pan, G. and D. Pei. 2005. The stem cell pluripotency factor NANOG activates transcription with two unusually potent subdomains at its C terminus. J. Biol. Chem. 280:1401-1407.
Rodda, D. J., J. L. Chew, L. H. Lim, Y. H. Loh, B. Wang, H. H. Ng, and P. Robson. 2005. Transcriptional regulation of nanog by OCT4 and SOX2. J. Biol. Chem. 280:24731-24737.
Rosner, M. H., M. A. Vigano, K. Ozato, P. M. Timmons, F. Poirier, P. W. Rigby, and L. M. Staudt. 1990. A POU-domain transcription factor in early stem cells and germ cells of the mammalian embryo. Nature 345:686-692.
Shi, W., H. Wang, G. Pan, Y. Geng, Y. Guo, and D. Pei. 2006. Regulation of the pluripotency marker Rex-1 by Nanog and Sox2. J. Biol. Chem. 281:23319-23325.
Suzuki, A., A. Raya, Y. Kawakami, M. Morita, T. Matsui, K. Nakashima, F. H. Gage, C. Rodriguez-Esteban, and J. C. Izpisua Belmonte. 2006. Nanog binds to Smad1 and blocks bone morphogenetic protein-induced differentiation of embryonic stem cells. Proc. Natl. Acad. Sci. U. S. A 103:10294-10299.
Tai, M. H., C. C. Chang, M. Kiupel, J. D. Webster, L. K. Olson, and J. E. Trosko. 2005. Oct4 expression in adult human stem cells: evidence in support of the stem cell theory of carcinogenesis. Carcinogenesis 26:495-502.
Tanaka, S., T. Kunath, A. K. Hadjantonakis, A. Nagy, and J. Rossant. 1998. Promotion of trophoblast stem cell proliferation by FGF4. Science 282:2072-2075.
Uchikawa, M., Y. Kamachi, and H. Kondoh. 1999. Two distinct subgroups of Group B Sox genes for transcriptional activators and repressors: their expression during embryonic organogenesis of the chicken. Mech. Dev. 84:103-120.
Uwanogho, D., M. Rex, E. J. Cartwright, G. Pearl, C. Healy, P. J. Scotting, and P. T. Sharpe. 1995. Embryonic expression of the chicken Sox2, Sox3 and Sox11 genes suggests an interactive role in neuronal development. Mech. Dev. 49:23-36.
van Eijk, M. J., M. A. van Rooijen, S. Modina, L. Scesi, G. Folkers, H. T. van Tol, M. M. Bevers, S. R. Fisher, H. A. Lewin, D. Rakacolli, C. Galli, C. de Vaureix, A. O. Trounson, C. L. Mummery, and F. Gandolfi. 1999. Molecular cloning, genetic mapping, and developmental expression of bovine POU5F1. Biol. Reprod. 60:1093-1103.
Vejlsted, M., H. Offenberg, F. Thorup, and P. Maddox-Hyttel. 2006. Confinement and clearance of OCT4 in the porcine embryo at stereomicroscopically defined stages around gastrulation. Mol. Reprod. Dev. 73:709-718.
Wang, J., S. Rao, J. Chu, X. Shen, D. N. Levasseur, T. W. Theunissen, and S. H. Orkin. 2006. A protein interaction network for pluripotency of embryonic stem cells. Nature 444:364-368.
28
Wang, R., J. Li, and N. Yashpal. 2004. Phenotypic analysis of c-Kit expression in epithelial monolayers derived from postnatal rat pancreatic islets. J. Endocrinol. 182:113-122.
Wood, H. B. and V. Episkopou. 1999. Comparative expression of the mouse Sox1, Sox2 and Sox3 genes from pre-gastrulation to early somite stages. Mech. Dev. 86:197-201.
Wu, Q., X. Chen, J. Zhang, Y. H. Loh, T. Y. Low, W. Zhang, W. Zhang, S. K. Sze, B. Lim, and H. H. Ng. 2006. Sall4 interacts with Nanog and co-occupies Nanog genomic sites in embryonic stem cells. J. Biol. Chem. 281:24090-24094.
Yang, Y., W. Liu, Z. L. Liu, X. X. Hu, and N. Li. 2004. Mapping NANOG to chromosome 5 in swine. Anim Genet. 35:411.
29
Figures and Tables Figure 1 Porcine Nanog Nucleotide and Amino Acid Sequence.
Overall transcript length is 1181 base pairs encoding a 304 amino acid protein. Start and stop codons at nucleotide positions 194 and 1106 respectively. Homeodomain and tryptophan repeats begin at amino acid positions 97 and 195 respectively and are shaded.
1 ATTTTGCTAG ATTGGGGTGG TTAGCTCCTG TTCTCGTAAG GGTGACTCAC TTCATCCCAT TTTTTGATAC TTTTAACACC 81 TGGAGGAGAT CTTCATGATT CTAAGCTCTT CTATCTAGAC ACTTAAGCCT GGACTTTTCC TACAATCCAG CTCTTTGGTG 1 M S V D P A C P Q S L L C P E A 161 GTTTTTTTTT TTTTCCCCCT TCTTCAACTC AACATGAGTG TGGATCCAGC TTGTCCCCAA AGCCTGCTTT GCCCCGAAGC 17 S I S S E S S P M P E V Y G P E E N Y A S L Q M S S 241 ATCCATTTCC AGCGAATCTT CACCAATGCC TGAGGTTTAT GGGCCTGAAG AAAATTATGC CTCCTTGCAG ATGTCATCTG 43 A E T L D T E T V S P L P S S M D L L I Q D S P D S S 321 CTGAGACCCT CGACACCGAG ACTGTCTCTC CTCTTCCTTC CTCCATGGAT CTGCTTATTC AGGACAGCCC TGATTCTTCC 70 T S P R V K P L P T S A E K S T E K E E K V P V K K Q 400 ACAAGCCCCA GAGTAAAACC ACTGCCCACA TCTGCAGAGA AGAGCACAGA GAAGGAGGAA AAGGTCCCAG TCAAGAAGCA 97 K I R T V F S Q T Q L C V L N D R F Q R Q K Y L S L 481 GAAGATCAGA ACTGTGTTCT CGCAGACCCA GCTCTGTGTC CTCAACGACA GATTTCAGAG GCAGAAGTAC CTCAGCCTCC 123 Q Q M Q E L S N I L N L S Y K Q V K T W F Q N Q R M K 561 AGCAGATGCA AGAACTTTCC AACATCCTGA ACCTTAGCTA CAAACAGGTT AAAACCTGGT TCCAGAACCA GCGAATGAAA 150 C K R W Q K N H W P R N S N S V I Q G S A S T E Y P G 641 TGTAAGAGGT GGCAGAAAAA CCACTGGCCA AGGAATAGCA ACAGTGTGAT TCAGGGCTCA GCCAGTACAG AATACCCGGG 177 F Y S Y H Q G C L V N A S G N L P V W G N Q S W S N 721 CTTCTATTCC TACCACCAAG GATGCCTGGT GAACGCTTCT GGAAACCTGC CCGTGTGGGG TAATCAGAGC TGGAGTAACC 203 P T W S N Q T W N S Q S W S N Q T W N S Q T W C P Q A 801 CAACCTGGAG CAACCAGACC TGGAACAGCC AGTCTTGGAG CAACCAAACC TGGAACAGCC AGACCTGGTG CCCCCAAGCC 230 W N N Q T W N S Q L N N Y V E E F L Q P Q L Q F Q Q N 881 TGGAATAACC AGACTTGGAA TAGCCAGCTC AACAACTATG TTGAGGAATT CCTGCAGCCC CAGCTCCAGT TTCAGCAAAA 257 S I S D L E A V L E T A G E N H N V I Q Q T S K Y C 961 TTCTATCAGT GATTTGGAGG CCGTCTTGGA AACTGCTGGG GAAAATCATA ATGTAATACA GCAGACTTCA AAGTACTGCG 283 G T Q Q Q I M D L F P N Y S M N I Q P E D M & 1041 GTACCCAGCA GCAAATCATG GATTTATTCC CAAATTACTC CATGAACATA CAGCCTGAAG ATATGTGACG ATCATTTTAT 1121 TTTTTTAAAA AATTTTATTG GAATATAGTT GATTTACAAA AAAAAAAAAA AAAAAAAAAA A
30
Figure 2 Porcine Nanog amino acid alignment with the Human, Bovine and Mouse proteins.
Upper case letters represent complete consensus (4/4), lower case represent partial (3/4). Homedomain and tryptophan repeats are highlighted in the consensus sequence.
Porcine 1 MSVDPACPQS LLCPE-ASIS SESSPMPEVY GPEENYASLQ MSSAETLDTE TVSPLPSSMD LLIQDSPDSS TSPRVKPLPT Human 1 MSVDPACPQS LPCFE-ASDC KESSPMPVIC GPEENYPSLQ MSSAEMPHTE TVSPLPSSMD LLIQDSPDSS TSPKGKQPTS Bovine 1 MSVGPACPQS LLGPE-ASNS RESSPMPE-- ---ESYVSLQ TSSADTLDTD TVSPLPSSMD LLIQDSPDSS TSPRVKPLSP Mouse 1 MSVGLPGPHS LPSSEEASNS GNASSMPAVF HP-ENYSCLQ GSATEMLCTE AASPRPSSED LPLQGSPDSS TSPKQKLSSP Consensus MSV pacPqS L E AS s esSpMP EnY sLQ Ssae l Te tvSPlPSSmD LliQdSPDSS TSP K
Porcine 80 SAEKSTEKEE –KVPVKKQKI RTVFSQTQLC VLNDRFQRQK YLSLQQMQEL SNILNLSYKQ VKTWFQNQRM KCKRWQKNHW Human 80 AEKSVAKKED –KVPVKKQKT RTVFSSTQLC VLNDRFQRQK YLSLQQMQEL SNILNLSYKQ VKTWFQNQRM KSKRWQKNNW Bovine 75 SVEESTEKEE –TVPVKKQKI RTVFSQTQLC VLNDRFQRQK YLSLQQMQEL SNILNLSYKQ VKTWFQNQRM KCKKWQKNNW Mouse 80 EADKGPEEEE NKVLARKQKM RTVFSQAQLC ALKDRFQKQK YLSLQQMQEL SSILNLSYKQ VKTWFQNQRM KCKRWQKNQW Consensus ekEe kVpvkKQK RTVFSqtQLC vLnDRFQrQK YLSLQQMQEL SnILNLSYKQ VKTWFQNQRM KcKrWQKN W
Porcine 159 PRNSNSVIQ- GSASTEYPGF YS–YHQGCLV NASGNLPVWG NQSWSNPTWS NQTW-----N SQSWSNQTWN SQTWCPQAWN Human 159 PKNSNGVTQ- KASAPTYPSL YSSYHQGCLV NPTGNLPMWS NQTWNNSTWS NQTQ-----N IQSWSNHSWN TQTWCTQSWN Bovine 154 PRNSNGMPQ- GPAMAEYPGF YS-YHQGCLV NSPGNLPMWG NQTWNNPTWS NQSW-----N SQSWSNHSWN SQAWCPQAWN Mouse 160 LKTSNGLIQK GSAPVEYPSI HCSYPQGYLV NASGSLSMWG SQTWTNPTWS SQTWTNPTWN NQTWTNPTWS SQAWTAQSWN Consensus p nSNg Q g a eYP ys YhQGcLV N GnLpmWg nQtW NpTWS nQtw N QsWsN Wn Sq Wc Q WN
Porcine 238 NQTWNS-QLN NYVEEFLQPQ LQFQQNS-IS DLEAVLETAG ENHNVIQQTS KYCGTQQQIM DLFPNYSMNI QPEDM Human 238 NQAWNS-PFY NCGEESLQSC MQFQPNSPAS DLEAALEAAG EGLNVIQQTT RYFSTPQT-M DLFLNYSMNM QPEDV Bovine 233 NQPWNN-QFN NYMEEFLQPG IQLQQNSPVC DLEATLGTAG ENYNVIQQTV KYFNSQQQIT DLFPNYPLNI QPEDL Mouse 240 GQPWNAAPLH NFGEDFLQPY VQLQQNFSAS DLEVNLEATR ESH------- AHFSTPQA-L ELFLNYSVTP PGEI Consensus nQ WN N EefLQp Q QqNs s DLEa Le ag E nviqqt yf t Q dLF NYs n qpEd
31
Figure 3 Porcine Nanog Promoter
Numbers at left are position relative to transcription start site and at right are overall. A putative ES specific enhancer and Oct/Sox element are highligted at position -259 and at -148. Transcription start site (+1) and start codon (+194) are also highlighted.
-453 GAAAAATGGA GCTAACATGT TTCTGCAGAA TAAGCCTGAA CTGGAGACCC AAAGGAGTCT 60
-393 CAGGTCAAGA AATTCATTGT CCCAGCGGGA GTTTCAGTCA CCGGAAATAG CCTCAGGAAC 120
-333 TGGAGGTGCA TCTTCCATTT GATCTGATTT TTTTTTTTTT TAATTTTTAA AAAAATTTTT 180
-273 TGCATCTTTG ATTTTAAAAA GTGGAAACAC GGTGGACCTG CAAGTAGTTC ACTGCGGGGT 240
-213 TTATTTTGTT TCCAGGTTCC ATGGTCCCAG TTCCCCACCC AGTCTGGGTT ACTCAGCAGC 300
-153 CCTCTCTTTG CATTACAATG GCCTTGGTGA GGCTGGCAGA CGGGATTAAC TGGGAATTCG 360
-93 CAAGGGTGTG TGTGGGCGTG GGGCTGCCAG GAGGGGCGGG CTTAAGTATG GTCGATCCTT 420
-33 CCTTATAAAT CTAGAGCCTC CAAAATTTTT CTCATTTTGC TAGATTGGGG TGGTTAGCTC 480
28 CTGTTCTCGT AAGGGTGACT CACTTCATCC CATTTTTTGA TACTTTTAAC ACCTGGAGGA 540
88 GATCTTCATG ATTCTAAGCT CTTCTATCTA GACACTTAAG CCTGGACTTT TCCTACAATC 600
148 CAGCTCTTTG GTGGTTTTTT TTTTTTTCCC CCTTCTTCAA CTCAACATGA GTGTGGATCC 660
208 AGCTTGTCCC CAAAGCCTGC TTTGCCCCGA AGCATCCATT TCCAGCGAAT CTTCACCAAT 720
268 GCCTGAGGTT TATGGGCCTG AAGAAAATTA TGCCTCCTTG CAGATGTCA 769
32
Figure 4 Porcine Nanog Sequence Alignments with Bovine, Human and Mouse
A region of high sequence similarity as described by Rodda et al., (2005). The Oct/Sox element is conserved except for the first base pair in the porcine. M1 is also involved in Nanog regulation but is uncharacterized.
M1 Oct/Sox Element Porcine -180 CCCACCCAGTCTGGGTTACTCAGCAGCCCTCTCTTTGCATTACAATGGCCTTGGTGAGGC Bovine CCCACCGGGTCTGGGTTACTCTGCAACTCT-CTTTTGCATTACAATGGCCTTGGTGAGAC Human CCCACCTAGTCTGGGTTACTCTGCAGCTA--CTTTTGCATTACAATGGCCTTGGTGAGAC Mouse CCCTCCCAGTCTGGGTCACCTTACAGCTT--CTTTTGCATTACAATGTCCATGGTGGACC Porcine -120 TGGCAGACGGGATTAACTGGGAATTCGCAAGGGTGTGT Bovine TGGCAGACGGGATTAACTGGGAATTCGCAAGGGTGTGT Human TGGTAGACGGGATTAACTGAGAATTCACAAGGGTGGGT Mouse CTGCAGGTGGGATTAACTGTGAATTCACAGGGCTGGTG
33
Figure 5 Partial Porcine Oct-4 Sequence
725 bp of the porcine Oct-4 coding sequence including 425 bp of coding sequence. Highlighted are the 59 amino acid POU homeodomain and the stop codon.
1 D N N E N L Q E I C K A E T L V Q A R 1 CTGACAACAA CGAGAATCTG CAGGAGATAT GCAAGGCAGA GACCCTCGTG CAGGCCCGGA 20 K R K R T S I E N R V R G N L E S M F L 61 AGAGAAAGCG GACAAGTATC GAGAACCGAG TGAGAGGCAA CCTGGAGAGC ATGTTCCTGC 40 Q C P K P T L Q Q I S H I A Q Q L G L E 121 AGTGCCCAAA GCCCACTCTG CAGCAGATCA GCCACATCGC CCAGCAGCTC GGGCTAGAGA 60 K D V V R V W F C N R R Q K G K R S S S 181 AGGATGTGGT CCGCGTGTGG TTCTGCAACC GTCGCCAGAA GGGCAAACGA TCAAGCAGTG 80 D Y S Q R E D F E A A G S P F P G G P V 241 ACTATTCGCA ACGAGAGGAT TTTGAGGCTG CTGGGTCTCC TTTCCCAGGG GGACCAGTAT 100 S F P L A P G P H F G T P G Y G G P H F 301 CCTTTCCTCT GGCGCCAGGG CCCCATTTTG GTACCCCAGG CTATGGGGGC CCTCACTTCA 120 T T L Y S S V P F P E G E A F P S V S V 361 CCACCCTGTA CTCCTCGGTC CCATTCCCTG AGGGTGAGGC CTTTCCCTCG GTGTCTGTCA 140 T P L G S P M H S N & 421 CCCCTCTGGG CTCCCCCATG CATTCAAACT GAGGTGCCTG CCCTTCCCAG GAGTGGGGGG 481 GGTGAGGAAG GGGTGAGCTA GGGAGAGAAG CCTGGGGTTT GTACCAGGGC TTTGGGATTA 541 AGTTCTTCAT TCACTAAGAA AGGAATTGGG AACACAAAGG GTGTGGGGGC AGGGAGTCTA 600 GGGGAACTGG TTGGAGGGAA GGTGAAGTTC AATGATGCTC TTGATTTTAA TCCCCACATC 661 ACTCATCACT TTGTTCTTAA ATAAAGAAGC CTGGGACCCA GAAAAAAAAA AAAAAAAAAA 721 AAAAA
34
Figure 6 Partial Porcine Sox-2 Sequence
861 bp of Sox-2 sequence including 733 bp of coding sequence. Highlighted are 37 residues of the HMG binding domain and stop codon.
1 L G A E W K L L S E T E K R P F I D E A 1 GCTGGGCGCC GAGTGGAAAC TTTTGTCGGA GACGGAGAAG CGGCCGTTCA TCGACGAGGC 21 K R L R A L H M K E H P D Y K Y R P R R 61 CAAGCGGCTG CGAGCGCTGC ACATGAAGGA GCACCCGGAT TATAAATACC GGCCCCGGCG 41 K T K T L M K K D K Y T L P G G L L A P 121 GAAAACCAAG ACGCTCATGA AGAAGGATAA GTACACACTG CCCGGAGGGC TGCTGGCCCC 61 G G N S M A S G V G V G A G L G A G V N 181 GGGAGGCAAC AGCATGGCGA GCGGGGTCGG GGTGGGCGCT GGCCTCGGCG CGGGCGTGAA 81 Q R M D S Y A H M N G W S N G S Y S M M 241 CCAGCGCATG GACAGCTACG CGCACATGAA TGGCTGGAGC AACGGCAGCT ACAGCATGAT 101 Q D Q L G Y P Q H P G L N A H S A A Q M 301 GCAGGACCAG CTGGGCTATC CGCAGCACCC GGGCCTCAAT GCGCACAGCG CGGCTCAGAT 121 Q P M H R Y D V S A L Q Y N S M T S S Q 361 GCAGCCCATG CACCGCTACG ACGTGAGCGC CCTGCAGTAC AACTCCATGA CCAGCTCGCA 141 T Y M N G S P T Y S M S Y S Q Q G T P G 421 GACCTACATG AACGGCTCGC CCACCTACAG CATGTCCTAC TCGCAGCAGG GCACCCCTGG 161 M A L G S M G S V V K S E A S S S P P V 481 CATGGCGCTC GGTTCCATGG GCTCAGTGGT CAAGTCCGAG GCGAGCTCCA GCCCCCCCGT 181 V T S S S H S R A P C Q A G D L R D M I 541 GGTTACCTCT TCTTCCCACT CCAGGGCGCC CTGCCAGGCC GGGGACCTAC GGGACATGAT 201 S M Y L P G A E V P E P A A P S R L H M 601 CAGCATGTAC CTCCCCGGCG CTGAGGTGCC AGAGCCCGCC GCCCCCAGCA GACTTCACAT 221 S Q H Y Q S G P V P G T A I N G T L P L 661 GTCCCAGCAC TACCAGAGCG GCCCGGTGCC CGGCACGGCC ATCAACGGTA CACTGCCTCT 241 S H M & 721 CTCTCACATG TGAGGGCCGG ACAGTGAACT GGAGGGGGCG GGGGGAGAAA ATTTTCAAAG 781 AAAAAGAGGG AAATGGGAGG AGAGTAAGAA ACAGTATGGA GAAAAACCCG GTACGCTTAA 841 AAAAAAAAAA AAAAAAAAAA A
35
Table 1 Adjusted Threshold Means for Nanog, Sox-2, and Oct-4 by Tissue.
Tissue Sample # Gene Meana Std. Error
D10 Conceptus 3 Nanog 21.59 0.74
Oct-4 21.05 0.74
Sox-2 21.20 0.87
D12 Conceputs 4 Nanog 21.76 0.64
Oct-4 21.27 0.64
Sox-2 21.61 0.75
D15 Disk 5 Nanog 20.94 0.57
Oct-4 20.95 0.57
Sox-2 15.92 0.67
D15 Distal 5 Nanog 24.56 0.57
Oct-4 23.90 0.57
Sox-2 25.57 0.67
D15 Proximal 3 Nanog 22.58 0.74
Oct-4 21.31 0.74
Sox-2 23.17 0.87
D17 Allantois 3 Nanog 21.75 0.74
Oct-4 23.54 0.74
Sox-2 22.27 0.87
D17 Disk 3 Nanog 21.08 0.74
Oct-4 22.84 0.74
Sox-2 15.09 0.87
D17 Distal 3 Nanog 24.21 0.74
Oct-4 23.83 0.74
Sox-2 24.08 0.87
D17 Proximal 2 Nanog 22.86 0.90
Oct-4 21.87 0.91
Sox-2 25.58 1.06
aNormalized to 18s ribosomal RNA
36
Table 1 continued Adjusted Threshold Means Nanog, Sox-2, and Oct-4 by Tissue
Tissue Sample # Gene Meana Std. Error D17 Whole 3 Nanog 21.96 0.74 Oct-4 21.63 0.74
Sox-2 16.85 0.87
D40 Endometrium 3 Nanog 21.75 0.74
Oct-4 26.60 0.74
Sox-2 26.07 0.87
D40 Liver 3 Nanog 19.78 0.74
Oct-4 22.25 0.74
Sox-2 19.91 0.87
D40 Myometrium 3 Nanog 18.38 0.74
Oct-4 26.99 0.74
Sox-2 25.21 0.87
D40 Placenta 3 Nanog 19.24 0.74
Oct-4 26.39 0.74
Sox-2 23.67 0.87
aNormalized to 18s ribosomal RNA
37
Figure 7 Relative Nanog Expression in the Early Porcine Conceptus, Fetal Liver, Placenta, and Maternal Endometrium and
Myometrium
Nanog
1.3 3.9 3.37.0 6.1 7.8 7.0
12.3 11.27.0
27.4
40.0
72.6
1.000.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
d15 distal
d17 distal
d15 proximal
d17 proximal
d17 allantois
d17 whole
d10conceptus
d12conceptus
d15 disk
d17 disk
endometruim
liver
placenta
myom
etrium
Rel
ativ
e Ex
pres
sion
y
Extraembryonic Embryonic Day 40
azab bc abc
c ccccd cd
c
de
de
e
yFold difference relative to the tissue with lowest expression (d15 distal). See Table 1. zTissues with different letters differ P < 0.05.
38
Figure 8 Relative Oct-4 Expression in the Early Porcine Conceptus, Fetal Liver, Placenta, and Maternal Endo- and
Myometrium
Oct-4
8.5 9.0
51.4
34.9
11.0
41.1
61.4
52.9
65.7
17.8
1.0 1.3 1.5
26.7
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
d15 distal
d17 distal
d15 proximal
d17 proximal
d17 allantois
d17 whole
d10 conceptus
d12 conceptus
d15 disc
d17 disc
myom
etrium
endometruim
placenta
liver
Rel
ativ
e Ex
pres
sion
y
bz bb
db
bcdb
bcb
cd
db
db
db
bcdb
ab
ab
ab
bcdb
Day 40EmbryonicExtraembryonic
yFold difference relative to the tissue with lowest expression (myometrium). See Table 1. zTissues with different letters differ P < 0.05.
39
Figure 9 Relative Sox-2 Expression in the Early Porcine Conceptus, Fetal Liver, Placenta, and Maternal Endo- and
Myometrium
Sox2
1.4 4.0 7.5 1.4 13.9
595.9
29.2 22.0
1134.6
2022.6
1.0 1.8 5.3 71.50.0
500.0
1000.0
1500.0
2000.0
2500.0
d15 distal
d17 distal
d15 proximal
d17 proximal
d17 allantois
d17 whole
d10 conceptus
d12 conceptus
d15 disc
d17 disc
endometruim
myom
etrium
placenta
liver
Rel
ativ
e Ex
pres
sion
y
Extraembryonic Embryonic Day 40
a e
f
f
f
abaz ababc abcdbcd cde de de
yFold difference relative to the tissue with lowest expression (endometrium). See Table 1. zTissues with different letters differ P < 0.05.
40
Figure 10 Northern Blot of Porcine Sox-2
Ten ug of total RNA from d-12 conceptus (A), d-15 disk (B), d-15 distal extraembryonic (C) and d-15 proximal extraembryonic (D) were probed for Sox-2 mRNA. Film was exposed for 1hr 30min. A band of ~2300 bp was observed in the d-15 disk (B).
2500
2000
A C DB2500
2000
A C DB
41
Figure 11 Northern Blots of Porcine β-Actin
Ten ug (A-D) and 20 ug (E-G) of total RNA from d-12 conceptus (A,G), d-15 disk (B), d-15 distal extraembryonic (C) d-15 proximal extraembryonic (D), d-17 disk (E), and allanotis (F) were probed for β-Actin. Filmed was exposed overnight. Two bands for β-Actin were observed (~ 1550 and 1750).
2000
1500
E F G2000
1500
E F G
2000
1500
E F G2000
1500
E F G
42
Figure 12 Western Blot of Porcine Nanog
Approximately 20 ug of protein, from porcine fibroblasts (A), mouse ES D3 cells (B), d-15 extraembryonic (C), and d-15 conceptus (D), were probed for Nanog. It is believed the 50 kDa band is Nanog.
[ ] [ ] [ ] [ ]
[ ] [ ] [ ]
[ ] [ ]
45
31
21
A B C D
[ ] [ ] [ ] [ ]
[ ] [ ] [ ]
[ ] [ ]
[ ] [ ] [ ] [ ]
[ ] [ ] [ ]
[ ] [ ]
45
31
21
A B C D
43
CHAPTER 3 - Timed Insemination of Beef Heifers using the 7-11
Synch Protocol
Abstract In Exp. 1, 179 yearling heifers were either fed melengestrol acetate (MGA; 7-11 Synch)
or given an intravaginal progesterone (P4)-releasing insert (CIDR; 7-11 CIDR) for 7 d.
Prostaglandin F2α (PG) was administered on the last day of MGA feeding or at CIDR removal
followed by the Cosynch protocol (GnRH – PG – GnRH) beginning 4 d after MGA withdrawal
or 2 d after CIDR removal. Heifers were fixed-time AI (TAI) 48 h after the second PG. Blood
samples were collected at d -10, d 1 (start of MGA feeding) and d 18 (second PG injection). In
Exp. 2, 298 yearling heifers were treated with the 7-11 Synch protocol or with the 7-11 Synch
protocol without the first GnRH injection (7 Synch) and TAI beginning 54 hr after PG. Blood
samples were collected at d -10 and d 1 in yr 1 and d -10, 1, 18 and at TAI in yr 2. In Exp. 1,
there was no difference between treatments in inducing ovulation in prepubertal heifers (94 vs
78; P = 0.21), the proportion of heifers that had luteal tissue on d 18 (87 vs 83%; P = 0.39) or
pregnancy rates to 48 hr TAI (47 vs 46%; P = 0.84) between 7-11 Synch and 7-11 CIDR
treatments respectively. In Exp. 2, the administration of the GnRH after MGA removal tended
(P = 0.07) to induce more prepuberal heifers to cycle (88 vs 61%) and increased (P < 0.01) the
proportion of heifers with luteal tissue on d 18 (88 vs 72%). Pregnancy rates for a 54 hr TAI
were higher (P < 0.01) for the 7-11 Synch treatment (55%) compared to 7 Synch (38%). We
conclude that there is no difference in pregnancy rates between MGA and CIDR when included
before the Cosynch protocol. However, the use of GnRH induces more prepubertal heifers to
ovulate and improves the proportion of heifers with luteal tissue at the PG injection which
increased pregnancy rates to a timed artificial insemination..
Introduction
Melengestrol acetate (MGA) has been included in artificial insemination protocols
because it is a cost effective and easy to administer progestogen that can induce prepubertal
heifers or anestrus cows to become cyclic. Long-term feeding (14 d) of MGA (0.5mg·animal-1·d-
44
1) has been effective in estrus synchronization of heifers (Brown et al., 1988; Jaeger et al., 1992)
and cows (Bader et al., 2005; Patterson et al., 1995; Stegner et al., 2004c; Stegner et al., 2004a).
Recent synchronization protocols using MGA have focused on reducing the length of MGA
feeding and use of GnRH to synchronize the follicular wave for a fixed-timed AI (TAI).
A high proportion of heifers exhibit estrus in response to short-term MGA feeding but
with lower fertility (Beal et al., 1988) (Chenault et al., 1990). Therefore, a program that
synchronized first-wave follicles after MGA feeding with GnRH was developed called 7-11
Synch (Kojima et al., 2000). This protocol has been used successfully in postpartum beef cows
by inseminating after observed estrus (Kojima et al., 2000; Stegner et al., 2004c) and at a fixed
time (Bader et al., 2005; Kojima et al., 2002; Kojima et al., 2003) but has not been reported in
beef heifers. The objectives of this study were to compare MGA and an intravaginal
progesterone (P4)-releasing insert (CIDR) using the 7-11 Synch protocol and the effect of GnRH
on prepubertal heifers to induce cyclicity and improve pregnancy rates to a fixed-time AI after
short-term MGA feeding.
Material and Methods
Experiment 1
Experimental Design.
Crossbred Angus heifers from two locations (CCR; n=51and CCU; n=79) and purebred
Angus, Hereford, and Simmental heifers (PBU; n=50) from a third location were randomly
assigned to two treatments (Figure13). The 7-11 Synch treated heifers were fed MGA (0.5
mg·animal-1·d-1 MGA 200 Premix, Pharmacia & Upjohn Company, Kalamazoo, MI) in a
sorghum grain carrier for 7 days beginning on d 1 followed by an injection of PGF2α (PG; 25 mg
i.m. of Lutalyse, Pharmacia & Upjohn Company) on d 7. An injection of GnRH (100 μg i.m. of
OvaCyst, IVX Animal Health, Inc., St. Joseph, MO) was given on d 11, followed by PG on d 18,
and GnRH at the time of insemination. Heifers assigned to 7-11 CIDR received an Eazi-Breed
CIDR (Pharmacia & Upjohn Company) beginning on d 3. On d 9, CIDRs were removed and
given PG, followed by GnRH on d 11, PG on d 18 and GnRH at the time of insemination.
AI and Pregnancy Determination
45
Heifers were TAI beginning 48 h after PG on d 18. The same two inseminators were
used for all locations. Two different sires were used at the CCR and CCU locations and 11 sires
were used at the PBU location. Purebred heifers were checked twice daily for 45 d after TAI and
were bred 12 h after the onset of estrus. Crossbred heifers were exposed to bulls for 60 to 80
days beginning 7 days after insemination. Conception rate to AI was determined by transrectal
ultrasonography (Aloka 500V with a 5.0-MHz linear array probe; Aloka, Wallingford, CT) 30 to
35 days after insemination. Final pregnancy status was determined 50-60 days after the natural
service breeding season by rectal palpation.
Blood Collection and Progesterone Concentrations
Blood samples were collected via coccygeal venipuncture on d -10, d 1, and d 18 to
determine heifer cyclicity and treatment response. Blood was allowed to clot overnight at 4oC
and serum was separated by centrifugation the following day. Serum was frozen at -20oC until
assays for P4 concentration by RIA were performed. Heifers with P4 concentrations > 1 ng/mL
either on d -10 or d 1 were considered to have obtained puberty before treatments. Heifers not
cycling before treatment but had P4 concentrations > 1 ng/mL at d 18 or conceived to TAI was
considered to have been induced to ovulate during treatments.
Experiment 2
Experimental Design
Experiments took place at two locations and over two years using crossbred Angus
heifers (CCU05; n=73 and CCU06; n=91) and purebred Angus, Hereford, and Simmental heifers
(PBU05; n=58 and PBU06; n=66). A third location (SF06) consisting of 21 purebred Angus
heifers was included in year 2. Beginning on d 1, all heifers in both treatments (Figure 14) were
fed MGA for 7 days and injected with PG on d 7. MGA was fed in a sorghum grain carrier at
locations 1 and 2 and in a grain pellet at location 3. Heifers randomly assigned to the 7-11 Synch
treatment received a GnRH injection on d 11, PG on d18, and GnRH at the time of breeding.
The 7 Synch treated heifers were given PG on d 18 and GnRH at the time of breeding.
AI and Pregnancy Determination
Timed insemination began 54 h after PGF injection. The same two AI technicians were
used both years and in all herds. In year one, eight sires and four sires were used at PBU05 and
46
CCU 05, respectively. In year two, five sires were used at PBU06, two sires at CCU06, and two
sires were used SF06. In herd one, heifers were heat checked twice daily for approximately 30 d
and inseminated 12 h from the onset of estrus and then exposed to bulls for another 30 d. In herd
two and three, heifers were exposed to bulls 7 d after the timed insemination for a 60 d breeding
season.
Blood Collection and Progesterone Concentrations
In year one, blood samples were collected via the coccygeal vein d -10 and d 1 to
determine heifer cyclicity before treatment. In year two, blood samples were collected on d -10,
d 1, d 18, and at the time of insemination. Blood was allowed to clot overnight at 4oC and serum
was separated by centrifugation the following day. Serum was frozen at -20oC until assays for
progesterone concentration by RIA were performed. Serum progesterone concentrations on the
day of breeding were determined for only those heifers who did not conceive to the timed
insemination. Heifers with progesterone concentrations > 1 ng/mL on d -10, d 1, or d 18 were
considered to have obtained puberty.
Semen Analysis
Semen analysis was done by the Andrology Laboratory at the Kansas State University
Veterinary Medical Teaching Hospital (Manhattan, KS) on 2 sires used in experiment 1. Semen
from eight sires used in experiment 2 was evaluated at the Bovine Andrology Lab (Penn
Veterinary Medicine, Kennett Square, PA) for sperm motility, sperm morphology, sperm
concentration, volume, and total sperm/dose. Results are presented in Table 10.
Statistical Analysis
Each experiment was analyzed by the Glimmix procedure of SAS (SAS Inst., Inc., Cary,
NC). Pretreatment cyclicity and overall cyclicity at d18 were analyzed using the model of
location, treatment, and location x treatment. Pretreatment cyclicity was included in the model
when proportion of heifers with P4 > 1ng/ml and actual P4 concentrations at d18 was analyzed.
The full model to predict pregnancy rate included treatment, cyclicity, and presence of CL at d18
as fixed effects and location, AI technician, and sire nested within location as random effects.
Significance of random effects was tested by likelihood ratio tests. Terms that were not
significant were dropped from the models.
47
Results
Experiment 1
Results from Exp. 1 are summarized in Table 2. There was no difference in the
proportion of cycling heifers at the beginning of treatment between treatments (P = 0.97) or
between locations (P = 0.18). Non-cycling heifers that were induced to ovulate were not
different (P = 0.21) between MGA (94%) and CIDR (78%) treated heifers. Proportion of heifers
with high P4 (≥ 1ng/mL) at d 18 was not different (P = 0.39) between 7-11 Synch and 7-11
CIDR treatments (87 vs 83%) but was different between locations (P = 0.04) (see Table 4). At
one location only 78% of the heifers had high P4 at d 18 compared to > 90% at the other 2
locations and the cycling heifers tended to be higher compared to the non-cycling heifers (87 vs
77%). Day 18 progesterone concentrations were significantly higher in the 7-11 Synch
compared to 7-11 CIDR (P < 0.01), was lower in the CCU herd (P < 0.01) when compared to the
other herds, and was higher in heifers that were cycling (P = 0.03) before treatment compared
with those that were not. For pregnancy rates to a timed AI, sire was a significant source of
variation (P = 0.04) but location (variance estimate = 0) and inseminator (P = 0.09) were not
therefore they were dropped in the final model. Pregnancy rates for 7-11 Synch (47%) and 7-11
CIDR (46%) were similar (P = 0.82). Pregnancy rates by herd and pubertal status are described
in Table 5.
Experiment 2
Results from Exp. 2 are summarized in Table 3. There was no difference in the
proportion of cycling heifers at the beginning of treatment between treatments or locations. The
percentage of prepubertal heifers induced to ovulate by d 18 were higher (P = 0.07) in the 7-11
Synch treatment (88%) compared to the 7 Synch treatment (61%). More 7-11 Synch treated
heifers (P < 0.01) had a CL (88%) on d 18 then did the 7 Synch treated heifers (72%). P4
concentrations at d 18 differed by treatment (P = 0.03) and herd (P < 0.02) and cycling status
before treatment (p=0.02) (see Table 6). Pregnancy rates to a TAI were higher (P < 0.01) in the
7-11 Synch treated heifers (55%) compared to 7 Synch (38%). Pregnancy rates by herd and
pubertal status are described in Table 7.
48
Discussion
Artificial insemination gives producers access to bulls that have high accuracy, are
superior in a particular trait(s), and/or they couldn’t afford otherwise. A successful
synchronization protocol should get more females to conceive earlier in the breeding season,
achieved in part by inducing pre-pubertal or anestrous females to cycle, and be economical and
efficient.
The 7-11 Synch is a synchronization protocol that uses short-term MGA feeding of 7
days followed by GnRH-PG to synchronize follicular growth and luteal regression (Kojima et
al., 2000). For a timed AI, GnRH is given at breeding so that 7-11 Synch is simply a Cosynch
protocol preceded by a presynchronization with a progestogen. Use of a progestogen in
synchronization induces ovulation in heifers (Jaeger et al., 1992) (Plugge et al., 1990) and cows
(Fike et al., 1997; Patterson et al., 1995), prevents a short luteal cycle from GnRH-induced
ovulations in postpartum cows (Thompson et al., 1999) and prevents early heats before or around
PG in a GnRH-PG-based protocol (Kojima et al., 2000).
Cyclicity
More than 95% of heifers had ovulated by d 18 after MGA or CIDR treatment when
followed by GnRH. Administration of GnRH after MGA withdrawal increased the number
prepubertal heifers to ovulate by 17%. Relative to other studies in cow, a high proportion of the
heifers were already cycling (> 75%) before treatment. An estrous response of 91% has been
observed in cows that were 90% anestrous at the beginning of the 7-11 synch treatment (Stegner
et al., 2004c). Breed, season, and nutritional status are other factors that can influence initiation
of puberty in heifers (Kinder et al., 1995).
In Exp. 1 and Exp. 2, prepubertal heifers in the 7-11 Synch treatment had pregnancy rates
4% and 10% higher than pubertal heifers but was not significant (p=0.72). Thus conception rates
are expected not to differ if a large proportion of heifers are not cycling at the beginning of
treatment.
Synchronization and Pregnancy Rates
Synchronization rates, the proportion heifers with high P4 at PG (d 18), were similar
between experiments (87 and 88%) for the 7-11 Synch treatment and for the 7-11 CIDR treated
49
heifers (83%). Our synchronization rates are similar to those reported for cows which have
ranged from 66 to 91% (Bader et al., 2005; Stegner et al., 2004c). In Exp. 2, 16% more heifers
were synchronized when given GnRH following MGA withdrawal. In two locations the
synchronization rate for 7-11 Synch was 96.4 and 90% and differed from the 7 Synch treatment
by 41 and 35 %. Kojima (Kojima et al., 2000) reported that only half of the females that did not
receive GnRH responded to the PG injection due to delayed ovulation after MGA withdrawal
and unresponsive early-developing CL.
Pregnancy rates to a timed AI using MGA or CIDR in the 7-11 Synch protocol were 47%
and 46% respectively which is lower than the pregnancy rates (> 60%) reported in postpartum
cows (Bader et al., 2005; Kojima et al., 2003). Average interval to estrus using 7-11 Synch has
been reported to be about 54 hr in heifers (Kojima et al., 2000) and from 52-64 hr in cows
(Kojima et al., 2000; Stegner et al., 2004c; Stegner et al., 2004b). Timed inseminations 60 hr
after PG have been suggested for cows (Bader et al., 2005; Kojima et al., 2003). Our lower
pregnancy rates maybe due to an earlier insemination time of 48 hr. In Exp. 2, a 54 hr TAI was
used and pregnancy rates ranged from 51 to 68% (55% overall) for 7-11 Synch. The 7 Synch
treatment had an average of 38%. Of the 7-11 Synch heifers that did not conceive to the TAI, 21
had high P4 at breeding; 15 had high P4 at d18 and 6 had low P4 at d18.
Differences in P4 concentrations can occur due to the hormonal environment under
which the dominant follicle develops. In 7-11 Synch, the first-wave dominant follicle is
developing under higher estradiol-17β concentrations and lower progesterone concentrations
compared to higher progesterone and lower estradiol-17β in a second-wave or mid-luteal follicle
(Stegner et al., 2004b). Since the dominant follicle in both treatments is a first wave follicle,
higher P4 concentrations may be due to a more mature CL induced by the d11 GnRH after MGA
withdrawal.
Since many of the response variables in our study had a binomial distribution such as
presence of luteal function on d 18 (yes/no) and pregnancy rate to TAI (pregnant/open), we used
the Glimmix Procedure in SAS which is a relatively new method for analysis of generalized
linear mixed models (GLMM). GLMM can be used when the response variable in not
necessarily normally distributed and can have any distribution in the exponential family which
includes binary, binomial, and Poisson distributions and when random effects are included in the
model assuming they are normal. The ability to include random effects offers an advantage over
50
other commonly used SAS procedures including Logistic and Genmod. In the absence of
random effects, the Glimmix procedure fits generalized linear models in the same manner as the
Genmod procedure. In the model for pregnancy rate, the variables location, inseminator, and sire
were treated as random effects likelihood ratio tests were used to test their significance in the
model.
51
Literature Cited
Bader, J. F., F. N. Kojima, D. J. Schafer, J. E. Stegner, M. R. Ellersieck, M. F. Smith, and D. J. Patterson. 2005. A comparison of progestin-based protocols to synchronize ovulation and facilitate fixed-time artificial insemination in postpartum beef cows. J. Anim Sci. 83:136-143.
Beal, W. E., J. R. Chenault, M. L. Day, and L. R. Corah. 1988. Variation in conception rates following synchronization of estrus with melengestrol acetate and prostaglandin F2 alpha. J. Anim Sci. 66:599-602.
Brown, L. N., K. G. Odde, M. E. King, D. G. Lefever, and C. J. Neubauer. 1988. Comparison of Melengestrol Acetate-Prostaglandin F(2)alpha to Syncro-Mate B for estrus synchronization in beef heifers. Theriogenology 30:1-12.
Chenault, J. R., J. F. McAllister, and C. W. Kasson. 1990. Synchronization of estrus with melengestrol acetate and prostaglandin F2 alpha in beef and dairy heifers. J. Anim Sci. 68:296-303.
Fike, K. E., M. L. Day, E. K. Inskeep, J. E. Kinder, P. E. Lewis, R. E. Short, and H. D. Hafs. 1997. Estrus and luteal function in suckled beef cows that were anestrous when treated with an intravaginal device containing progesterone with or without a subsequent injection of estradiol benzoate. J. Anim Sci. 75:2009-2015.
Jaeger, J. R., J. C. Whittier, L. R. Corah, J. C. Meiske, K. C. Olson, and D. J. Patterson. 1992. Reproductive response of yearling beef heifers to a melengestrol acetate-prostaglandin F2 alpha estrus synchronization system. J. Anim Sci. 70:2622-2627.
Kinder, J. E., E. G. Bergfeld, M. E. Wehrman, K. E. Peters, and F. N. Kojima. 1995. Endocrine basis for puberty in heifers and ewes. J. Reprod. Fertil. Suppl 49:393-407.
Kojima, F. N., B. E. Salfen, J. F. Bader, W. A. Ricke, M. C. Lucy, M. F. Smith, and D. J. Patterson. 2000. Development of an estrus synchronization protocol for beef cattle with short-term feeding of melengestrol acetate: 7-11 synch. J. Anim Sci. 78:2186-2191.
Kojima, F. N., J. E. Stegner, J. F. Bader, D. J. Schafer, R. L. Eakins, M. F. Smith, and D. J. Patterson. 2003. A comparison of two fixed-time AI programs for postpartum beef cows. J. Anim Sci. 81:50.
Kojima, F. N., J. E. Stegner, B. E. Salfen, R. L. Eakins, M. F. Smith, and D. J. Patterson. 2002. A fixed-time AI program for beef cows with 7-11 Synch. J. Anim Sci. 80:128.
Patterson, D. J., J. B. Hall, N. W. Bradley, K. K. Schillo, B. L. Woods, and J. M. Kearnan. 1995. Improved synchrony, conception rate, and fecundity in postpartum suckled beef cows fed melengestrol acetate prior to prostaglandin F2 alpha. J. Anim Sci. 73:954-959.
52
Plugge, B. L., G. H. Deutscher, M. K. Nielsen, and S. K. Johnson. 1990. Melengestrol acetate (MGA) and prostaglandin for estrous induction and synchonization in peripuberal beef heifers. Prof. Anim. Sci. 6:24.
Stegner, J. E., J. F. Bader, F. N. Kojima, M. R. Ellersieck, M. F. Smith, and D. J. Patterson. 2004a. Fixed-time artificial insemination of postpartum beef cows at 72 or 80 h after treatment with the MGA Select protocol. Theriogenology 61:1299-1305.
Stegner, J. E., F. N. Kojima, J. F. Bader, M. C. Lucy, M. R. Ellersieck, M. F. Smith, and D. J. Patterson. 2004b. Follicular dynamics and steroid profiles in cows during and after treatment with progestin-based protocols for synchronization of estrus. J. Anim Sci. 82:1022-1028.
Stegner, J. E., F. N. Kojima, M. R. Ellersieck, M. C. Lucy, M. F. Smith, and D. J. Patterson. 2004c. A comparison of progestin-based protocols to synchronize estrus in postpartum beef cows. J. Anim Sci. 82:1016-1021.
Thompson, K. E., J. S. Stevenson, G. C. Lamb, D. M. Grieger, and C. A. Loest. 1999. Follicular, hormonal, and pregnancy responses of early postpartum suckled beef cows to GnRH, norgestomet, and prostaglandin F2alpha. J. Anim Sci. 77:1823-1832.
53
Figures and Tables
7-11 Synch
1 7 11 18
PG GnRH
48 hrsTimed AI
GnRH
PG
7-11 CIDR
CIDR
3 9 18
PG GnRH
48 hrsTimed AI
PG
MGA
GnRH
11
7-11 Synch
1 7 11 18
PG GnRH
48 hrsTimed AI
GnRH
PG
7-11 CIDR
CIDR
3 9 18
PG GnRH
48 hrsTimed AI
PG
MGA
GnRH
11
Figure 13. Experiment 1 treatment schedule for heifers assigned to the 7-11 Synch and 7-
11 CIDR protocols.
Heifers assigned to the 7-11 Synch treatment were fed melengestrol acetate (MGA) for 7 d beginning on d 1 and injected with PGF2α (PG) on day of MGA withdrawal (d 7). The Cosynch protocol followed 4 d later; a GnRH injection (d 11), PG on d 18, and GnRH at breeding 48 h after PG. The 7-11 CIDR treatment began on d 3 with insertion of a CIDR for 7 d and a PG injection at CIDR removal. The Cosynch protocol began 2 d after CIDR removal (d 11).
54
7-11 Synch
1 7 11 18
PG GnRH
54 hrsTimed AI
GnRH
PG
7 Synch
MGA
1 7 18
PG GnRH
54 hrsTimed AI
PG
MGA
7-11 Synch
1 7 11 18
PG GnRH
54 hrsTimed AI
GnRH
PG
7 Synch
MGA
1 7 18
PG GnRH
54 hrsTimed AI
PG
MGA
Figure 14. Experiment 1 treatment schedule for heifers assigned to the 7-11 Synch and 7 Synch protocols.
Heifers assigned to the 7-11 Synch treatment were fed melengestrol acetate (MGA) for 7 d beginning on d 1 and injected with PGF2α (PG) on day of MGA withdrawal (d 7). The Cosynch protocol followed 4 d later; a GnRH injection (d 11), PG on d 18, and GnRH at breeding 48 h after PG. In the 7 Synch treatment, the d 11 GnRH injection is omitted.
55
Table 2 Comparison of 7-11 Synch using MGA (7-11 Synch) or CIDR (7-11 CIDR) on
different reproductive traits of heifers (Exp. 1)
Treatmenta
Item 7-11 Synch 7-11 CIDR
% (total No. of heifers) Cycling before initiation of treatmentw 79.8 (89) 78.9 (90) Prepubertal heifers induced to cycle by d 18x 94.4 (18) 78.9 (19) High progesterone on d 18 before PGy 87.5 (88) 83.2 (89) Pregnancy rates to TAI Overallz 47.1 (89) 46.6 (90) Cycling Status Pubertal 46.5 (71) 52.1 (71) Prepubertal 50.0 (18) 26.3 (19) Pregnancy rate at end of breeding season 92.1 (89) 91.1 (90) a7-11 Synch: beginning on d 1, melengestrol acetate (MGA) was fed for 7 d followed by injections of PGF2α (PG) on day of MGA withdrawal, GnRH on d 11, PG on d 18 and GnRH at breeding. 7-11 CIDR: insertion of CIDR for 7 d beginning on d 3. PG was injected at CIDR removal and GnRH was injected 2 d later (d11), PG at d 18, and GnRH at breeding. Timed inseminations began 48 h after PG. wTreatment difference P = 0.97 xTreatment difference P = 0.21 yTreatment difference P = 0.39 zTreatment difference P = 0.82
56
Table 3 Comparison of 7-11 Synch with (7-11 Synch) or without (7 Synch) GnRH on d 11 of treatment on different reproductive traits of heifers (Exp. 2)
Treatmenta
Item 7-11 Synch 7 Synch
% (total No. of heifers) Cycling before initiation of treatment 75.1 (149) 73.8 (149) Prepubertal heifers induced to cycle by d 18 88.2 (17) 61.9x (21) High progesterone on d 18 before PG 88.1 (84) 72.9y (85) Pregnancy rates to TAI Overall 55.3 (150) 38.0z (150) Cycling Status Pubertal 52.6 (112) 39.0 (110) Prepubertal 62.1 (37) 35.9 (39) Pregnancy rate at end of breeding season 87.5 (144) 84.2 (146) a7-11 Synch: beginning on d 1, melengestrol acetate (MGA) was fed for 7 d followed by injections of PGF2α (PG) on day of MGA withdrawal, GnRH on d 11, PG on d 18 and GnRH at breeding. 7-11 Synch: the GnRH injection at d 11 was omitted. Timed inseminations began 54 h after PG. xDifferent (P = 0.07) from 7-11 Synch yDifferent (P < 0.01) from 7-11 Synch zDifferent (P < 0.01) from 7-11 Synch
57
Table 4 Puberty status before treatment, overall puberty status after treatment, heifers with serum progesterone (P4) concentrations ≥1 ng/ml, and d 18 serum progesterone concentration of heifers by location
Heifer cyclicity1 Cyclicity at d182 CL present at d18 P4 concentrations at d18
% (total # of heifers) % (total # of heifers) % (total # of heifers) (ng/mL ± SE) Overall
7-11 Synch 79.8 (89) 98.8 (89) 87.5 (88) 2.75 ± 0.16x
7-11 CIDR 78.9 (90) 95.5 (90) 83.2 (89) 2.20 ± 0.15w
Combined 79.3 (179) 96.0 (179) 85.3 (177)
CCR
7-11 Synch 76.0 (25) 100 (25) 87.5 (24) 2.92 ± 0.27
7-11 CIDR 84.6 (26) 100 (26) 96.0 (25) 2.75 ± 0.28
Combined 80.4 (51) 100 (51) 91.8 (49) 2.82 ± 0.20y
CCU
7-11 Synch 82.1 (39) 97.4 (39) 84.6 (39) 1.84 ± 0.22
7-11 CIDR 87.2 (39) 94.8 (39) 71.8 (39) 1.33 ± 0.23
Combined 84.6 (78) 96.1 (78) 78.2 (78) 1.57 ± 0.17z
PBU
7-11 Synch 80.0 (25) 100 (25) 92.0 (25) 3.53 ± 0.27
7-11 CIDR 60.0 (25) 92.0 (25) 88.0 (25) 2.55 ± 0.26
Combined 70.0 (50) 96.0 (50) 90.0 (50) 3.03 ± 0.19y aPercentage of heifers that had progesterone serum concentrations ≥1 ng/mL in one of the two blood samples collected before treatment. bPercentage of heifers that was cyclic before treatement, had progesterone serum concentrations ≥1 ng/mL at d 18 or conceived to timed insemination. w,xTreatment means differ, P < 0.01 y,zLocation means differ, P < 0.01
58
Table 5 Pregnancy rates to 48 hr timed AI
Overall Pubertal Prepubertal
Pregnancy Rate at the end of breeding season
% (total # of heifers) % (total # of heifers) % (total # of heifers) % (total # of heifers) Overall
7-11 Synch 47.1 (89) 46.5 (71) 50.0 (18)
7-11 CIDR 46.6 (90) 52.1 (71) 26.3 (19)
Combined 46.9 (179) 49.3 (142) 37.8 (37)
CCR
7-11 Synch 48.0 (25) 42.1 (19) 60.0 (6) 100 (25)
7-11 CIDR 42.3 (26) 50.0 (22) 0.0 (4) 92.3 (26)
Combined 45.0 (51) 46.3 (41) 40.0 (10) 96.0 (51)
CCU
7-11 Synch 56.4 (39) 59.4 (32) 42.9 (7) 92.3 (39)
7-11 CIDR 48.7 (39) 50.0 (34) 40.0 (5) 92.3 (39)
Combined 52.5 (78) 54.5 (66) 41.7 (12) 92.3 (78)
PBU
7-11 Synch 32.0 (25) 30.0 (20) 40.0 (5) 83.0 (25)
7-11 CIDR 48.0 (25) 60.0 (15) 30.0 (10) 88.0 (25)
Combined 40.0 (50) 42.9 (35) 33.3 (15)
59
Table 6 Puberty status before treatment, overall puberty status after treatment, heifers with serum progesterone (P4) concentrations ≥1 ng/ml, and d 18 serum progesterone concentration of heifers by location
Pretreatment cyclicity1 Cyclicity at d18b P4 ≥1 ng/ml at d18 P4 concentrations at d18
% (total # of heifers) (ng/mL ± SE) Overall
7-11 Synch 75.1 (149) 97.6 (84) 88.1 (84) 2.91 ± 0.30w
7 Synch 73.8 (149) 90.5 (85) 72.9 (85) 2.09 ± 0.29x
Combined 74.5 (298) 94.0 (169) 80.4 (169)
CCU06
7-11 Synch 72.9 (37) - -
7 Synch 66.7 (36) - -
Combined 69.9 (73) - -
CCU07
7-11 Synch 82.6 (46) 95.6 (46) 82.6s (46) 3.05 ± 0.33
7 Synch 75.5 (45) 97.7 (45) 88.8s (45) 3.21 ± 0.33
Combined 79.1 (91) 96.7 (91) 85.7 (91) 3.13 ± 0.25u
PBU06
7-11 Synch 64.2 (28) - -
7 Synch 78.5 (28) - -
Combined 71.4 (56) - -
PBU07
7-11 Synch 71.4 (28) 100 (28) 96.4s (28) 3.24 ± 0.40
7 Synch 65.5 (29) 75.8 (29) 55.1t (29) 1.95 ± 0.39
Combined 68.4 (57) 87.7 (57) 75.4 (57) 2.59 ± 0.29uv
SF07
7-11 Synch 90.0 (10) 100 (10) 90.0st (10) 2.44 ±0.68
7 Synch 100 (11) 100 (11) 54.5t (11) 1.10 ± 0.66
Combined 95.2 (21) 100 (21) 71.4 (21) 1.77 ± 0.49v
Cycling
Yes 2.96 ± 0.21y
No 2.03 ± 0.37z aPercentage of heifers that had progesterone serum concentrations ≥1 ng/mL in one of the two blood samples collected before treatment. bPercentage of heifers that had progesterone serum concentrations ≥1 ng/mL before treatment, at d 18, or conceived to timed insemination. s,tTreatment means within location differ, P<0.01 u,vLocation means differ, P<0.05 w,xTreatment means differ, P<0.05 y,zCycling means differ, P<0.05
60
Table 7 Pregnancy rates to 54 hr timed AI
Overall Pubertal Prepubertal Pregnancy Rate at the end of breeding season
% (total # of heifers) % (total # of heifers) % (total # of heifers) % (total # of heifers) Overall
7-11 Synch 55.3y (150) 52.6 (112) 62.1 (37) 87.5 (144)
7 Synch 38.0z (150) 39.0 (110) 35.9 (39) 84.2 (146)
Combined 46.6 (300) 45.9 (222) 48.6 (76) 85.8 (290)
CCU06
7-11 Synch 51.3 (37) 40.7 (27) 80.0 (10) 86.4 (37)
7 Synch 52.7 (36) 66.6 (24) 25.0 (12) 80.5 (36)
Combined 52.0 (73) 52.9 (51) 50.0 (22) 83.5 (73)
CCU07
7-11 Synch 56.5 (46) 60.5 (38) 37.5 (8) 91.3 (46)
7 Synch 33.3 (45) 26.4 (34) 54.5 (11) 77.7 (45)
Combined 45.0 (91) 44.4 (72) 47.3 (19) 84.6 (91)
PBU06
7-11 Synch 68.9 (29) 66.6 (18) 70.0 (10) 82.7 (29)
7 Synch 44.8 (29) 40.9 (22) 66.6 (6) 89.2 (28)
Combined 56.9 (58) 52.5 (40) 68.7 (16) 85.9 (57)
PBU07
7-11 Synch 53.5 (28) 55.0 (20) 50.0 (8) 91.3 (23)
7 Synch 24.1 (29) 31.5 (19) 10.0 (10) 96.1 (26)
Combined 38.6 (57) 43.5 (39) 27.7 (18) 93.8 (49)
SF07
7-11 Synch 30.0 (10) 22.2 (9) 100.0 (1) 77.7 (9)
7 Synch 27.2 (11) 27.2 (11) - 81.8 (11)
Combined 28.5 (21) 25.0 (20) 100 (1) 80.0 (20)
61
Table 8 Pregnancy rate of heifers with low P4 on d 18
2005 2007
% (total #) % (total #) Overall Overall
7-11 Synch 63.6 (11) 7-11 Synch 40.0 (10)
7-11 CIDR 26.7 (15) 7 Synch 8.6 (23)
Combined 42.3 (26) Combined 18.1 (33)
CCR CCU07
7-11 Synch 66.6 (3) 7-11 Synch 25.0 (8)
7-11 CIDR 0.0 (1) 7 Synch 20.0 (5)
Combined 50.0 (4) Combined 23.0 (13)
CCU PBU07
7-11 Synch 66.6 (6) 7-11 Synch 100 (1)
7-11 CIDR 36.7 (11) 7 Synch 7.6 (13)
Combined 47.1 (17) Combined 14.2 (14)
PBU SF07
7-11 Synch 50.0 (2) 7-11 Synch 100 (1)
7-11 CIDR 0.0 (3) 7 Synch 0.0 (5)
Combined 20.0 (5) Combined 16.6 (6)
62
Table 9 P4 status of heifers that did not conceive Day 18 P4 Breeding P4
High (Total #)
Low (Total #)
High 6 28 7-11 Synch
Low 2 3
High 15 24 7 Synch
Low 6 15
63
Table 10 Semen Analysis
Conception Rate
Sire 0 hr Motility
(%) 2 hr Motility
(%) Normal Morphology
(%) Intact Acrosomes
(%) % No. Bingo1,3 48 10 75 77 35.9 (14/39)
Sleep Easy1,3 38 48 68 86 69.2 (27/39)
Bingo2,4 9 - 55 69 37.5 (9/24)
Paramount2,4 32 - 57 68 76.9 (10/13)
Sleep Easy2,4 58 - 80 90 50.0 (12/24)
Traveler2,4 44 - 72 93 58.3 (7/12)
Conservative2
,6 74 - 69 87 60.0 (6/10)
Domino2,6 40 - 47 66 14.3 (1/7)
Mo Better2,6 25 - 38 64 57.1 (4/7)
New Level2,5 48 - 61 87 50.0 (23/46)
1. Analysis by Andrology Laboratory, Veterinary Medical Teaching Hospital, Kansas State University, Manhattan Kansas. 2. Analysis by Reference Andrology Laboratory, Penn Veterinary Medicine, Kennett Square Pennslyvania. 3. 2005 Cow-calf Unit 4. 2006 Cow-calf Unit 5. 2007 Cow-calf Unit 6. 2007 Purebred Unit